Pyrazolopyrimidine PI3K inhibitor compounds and methods of use

ABSTRACT

Compounds of Formula I, and including stereoisomers, geometric isomers, tautomers, solvates, metabolites and pharmaceutically acceptable salts thereof, are useful for inhibiting lipid kinases including p110 alpha and other isoforms of PI3K, and for treating disorders such as cancer mediated by lipid kinases. Methods of using compounds of Formula I for in vitro, in situ, and in vivo diagnosis, prevention or treatment of such disorders in mammalian cells, or associated pathological conditions, are disclosed.

This application claims priority to U.S. Provisional Ser. No. 61/024,813filed on 30 Jan. 2008, the entire contents of which are incorporated byreference.

FIELD OF THE INVENTION

The invention relates generally to compounds with anti-cancer activityand more specifically to compounds which inhibit PI3 kinase activity.The invention also relates to methods of using the compounds for invitro, in situ, and in vivo diagnosis or treatment of mammalian cells,or associated pathological conditions.

BACKGROUND OF THE INVENTION

Phosphatidylinositol (hereinafter abbreviated as “PI”) is one of anumber of phospholipids found in cell membranes. In recent years it hasbecome clear that PI plays an important role in intracellular signaltransduction. Cell signaling via 3′-phosphorylated phosphoinositides hasbeen implicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity(Rameh et al (1999) J. Biol Chem, 274:8347-8350). The enzyme responsiblefor generating these phosphorylated signaling products,phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K),was originally identified as an activity associated with viraloncoproteins and growth factor receptor tyrosine kinases thatphosphorylate phosphatidylinositol (PI) and its phosphorylatedderivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al(1992) Trends Cell Biol 2:358-60).

Phosphoinositide 3-kinases (PI3K) are lipid kinases that phosphorylatelipids at the 3-hydroxyl residue of an inositol ring (Whitman et al(1988) Nature, 332:664). The 3-phosphorylated phospholipids (PIP3s)generated by PI3-kinases act as second messengers recruiting kinaseswith lipid binding domains (including plekstrin homology (PH) regions),such as Akt and phosphoinositide-dependent kinase-1 (PDK1). Binding ofAkt to membrane PIP3s causes the translocation of Akt to the plasmamembrane, bringing Akt into contact with PDK1, which is responsible foractivating Akt. The tumor-suppressor phosphatase, PTEN, dephosphorylatesPIP3 and therefore acts as a negative regulator of Akt activation. ThePI3-kinases Akt and PDK1 are important in the regulation of manycellular processes including cell cycle regulation, proliferation,survival, apoptosis and motility and are significant components of themolecular mechanisms of diseases such as cancer, diabetes and immuneinflammation (Vivanco et al (2002) Nature Rev. Cancer 2:489; Phillips etal (1998) Cancer 83:41).

The main PI3-kinase isoform in cancer is the Class I PI3-kinase, p110α(alpha) (U.S. Pat. No. 5,824,492; U.S. Pat. No. 5,846,824; U.S. Pat. No.6,274,327). Other isoforms are implicated in cardiovascular andimmune-inflammatory disease (Workman P (2004) Biochem Soc Trans32:393-396; Patel et al (2004) Proceedings of the American Associationof Cancer Research (Abstract LB-247) 95th Annual Meeting, March 27-31,Orlando, Fla., USA; Ahmadi K and Waterfield M D (2004) Encyclopedia ofBiological Chemistry (Lennarz W J, Lane M D eds) Elsevier/AcademicPress).

The PI3 kinase/Akt/PTEN pathway is an attractive target for cancer drugdevelopment since such agents would be expected to inhibitproliferation, reverse the repression of apoptosis and surmountresistance to cytotoxic agents in cancer cells. PI3 kinase inhibitorshave been reported (Yaguchi et al (2006) Jour. of the Nat. Cancer Inst.98(8):545-556; U.S. Pat. No. 7,173,029; U.S. Pat. No. 7,037,915; U.S.Pat. No. 6,608,056; U.S. Pat. No. 6,608,053; U.S. Pat. No. 6,838,457;U.S. Pat. No. 6,770,641; U.S. Pat. No. 6,653,320; U.S. Pat. No.6,403,588; U.S. Pat. No. 6,703,414; WO 97/15658; WO 2006/046031; WO2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP08176070).

Certain thienopyrimidine compounds have p110 alpha binding, PI3 kinaseinhibitory activity and inhibit the growth of cancer cells (WO2006/046031; WO 2007/122410; WO 2007/127183; WO 2007/129161; U.S. Ser.No. 11/951,189, “PHOSPHOINOSITIDE 3-KINASE INHIBITOR COMPOUNDS ANDMETHODS OF USE”, Castanedo et al, Filing Date Dec. 5, 2007; U.S. Ser.No. 11/951,203, “PHOSPHOINOSITIDE 3-KINASE INHIBITOR COMPOUNDS ANDMETHODS OF USE”, Bayliss et al, Filing Date Dec. 5, 2007).

SUMMARY OF THE INVENTION

The invention relates generally to pyrazolo[3,4-d]pyrimidines compoundsof Formula I with anti-cancer activity, and more specifically with PI3kinase inhibitory activity. Certain hyperproliferative disorders arecharacterized by the modulation of PI3 kinase function, for example bymutations or overexpression of the proteins. Accordingly, the compoundsof the invention may be useful in the treatment of hyperproliferativedisorders such as cancer. The compounds may inhibit tumor growth inmammals and may be useful for treating human cancer patients.

The invention also relates to methods of using thepyrazolo[3,4-d]pyrimidines compounds of Formula I for in vitro, in situ,and in vivo diagnosis or treatment of mammalian cells, organisms, orassociated pathological conditions.

Formula I compounds include:

and stereoisomers, geometric isomers, tautomers, or pharmaceuticallyacceptable salts thereof. The various substituents R¹, R², R³, R⁴ are asdefined herein.

Another aspect of the invention provides a pharmaceutical compositioncomprising a pyrazolo[3,4-d]pyrimidine compound of Formula I and apharmaceutically acceptable carrier. The pharmaceutical composition mayfurther comprise one or more additional therapeutic agent.

Another aspect of the invention provides methods of inhibiting PI3kinase activity, comprising contacting a PI3 kinase with an effectiveinhibitory amount of a compound of Formula I, or a stereoisomer,geometric isomer, tautomer, or pharmaceutically acceptable salt thereof.

Another aspect of the invention provides methods of preventing ortreating a hyperproliferative disease or disorder modulated by PI3kinases, comprising administering to a mammal in need of such treatmentan effective amount of a compound of Formula I, or a stereoisomer,geometric isomer, tautomer, or pharmaceutically acceptable salt thereof.Examples of such hyperproliferative disease or disorder include, but arenot limited to, cancer.

Another aspect of the invention provides methods of preventing ortreating a hyperproliferative disorder, comprising administering to amammal in need of such treatment an effective amount of a compound ofFormula I, or a stereoisomer, geometric isomer, tautomer, orpharmaceutically acceptable salt thereof, alone or in combination withone or more additional compounds having anti-hyperproliferativeproperties.

In a further aspect the present invention provides a method of using acompound of this invention to treat a hyperproliferative disease orcondition modulated by PI3 kinase in a mammal.

An additional aspect of the invention is the use of a compound of thisinvention in the preparation of a medicament for the treatment orprevention of a disease or condition modulated by PI3 kinase in amammal.

Another aspect of the invention includes kits comprising a compound ofFormula I, or a stereoisomer, geometric isomer, tautomer, solvate,metabolite, or pharmaceutically acceptable salt thereof, a container,and optionally a package insert or label indicating a treatment.

Another aspect of the invention includes methods of preparing, methodsof separating, and methods of purifying compounds of Formula I.

Another aspect of the invention includes novel intermediates useful forpreparing Formula I compounds.

Additional advantages and novel features of this invention shall be setforth in part in the description that follows, and in part will becomeapparent to those skilled in the art upon examination of the followingspecification or may be learned by the practice of the invention. Theadvantages of the invention may be realized and attained by means of theinstrumentalities, combinations, compositions, and methods particularlypointed out in the appended claims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.

DEFINITIONS

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁-C₈), orone to six carbon atoms (C₁-C₆). Examples of alkyl groups include, butare not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu,i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkylene radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkylene radical is one to eight carbon atoms(C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples of alkylene groupsinclude, but are not limited to, methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted, and includesradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations. Examples include, but are not limited to,ethylenyl or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and the like.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted, and includesradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations. Examples include, but are not limited to,ethylenylene or vinylene (—CH═CH—), allyl (—CH₂CH═CH—), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted. Examples include, but are notlimited to, ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and thelike.

The term “alkynylene” refers to a linear or branched divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp triple bond, whereinthe alkynyl radical may be optionally. Examples include, but are notlimited to, ethynylene (—C≡C—), propynylene (propargylene, —CH₂C≡C—),and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo[4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo[5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted.

“Arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of two hydrogen atom from a twocarbon atoms of a parent aromatic ring system. Some arylene groups arerepresented in the exemplary structures as “Ar”. Arylene includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typicalarylene groups include, but are not limited to, radicals derived frombenzene (phenylene), substituted benzenes, naphthalene, anthracene,biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene,1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionallysubstituted

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted, for example, with oxo (═O),mercapto, or amino, etc. A heterocycle may be a monocycle having 3 to 7ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected fromN, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbonatoms and 1 to 6 heteroatoms selected from N, O, P, and S), for example:a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl 1H-benzo[d]imidazol-2(3H)-one-5-yl, and N-pyridyl ureas.Spiro moieties are also included within the scope of this definition.Examples of a heterocyclic group substituted with one or more oxo (═O)moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5 to about 20 ring atoms, containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur.Examples of heteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups areoptionally substituted.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

The term “monocyclic heteroaryl” refers to a five- or six-membered,unsubstituted or substituted, monocyclic heteroaryl radical whichcontains 1, 2, 3 or 4 ring heteroatoms independently selected from N, Oand S. The monocyclic heteroaryl may be attached to the C-4 and C-6positions of the pyrimidine ring according to Formula I at any carbon(carbon-linked) atom of the monocyclic heteroaryl R³ group. Monocyclicheteroaryl radicals include, but are not limited to: 2-pyridyl,3-pyridyl, 4-pyridyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-imidazolyl, 4-imidazolyl, 3-pyrazolyl, 4-pyrazolyl, 2-pyrrolyl,3-pyrrolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 2-pyrimidinyl, 5-pyrimidinyl,6-pyrimidinyl, 2-pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 3-triazolyl, 1-triazolyl,5-tetrazolyl, 1-tetrazolyl, and 2-tetrazolyl. Monocyclic heteroaryls areoptionally substituted.

“Fused bicyclic C₄-C₂₀ heterocyclyl” and “Fused bicyclic C₁-C₂₀heteroaryl” containing one or more heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur, differ only by their aromaticcharacter, and have two rings fused together, i.e. share a common bond.Fused bicyclic heterocyclyl and heteroaryl radicals may be attached tothe C-4 and C-6 positions of the pyrimidine ring according to Formula Iat any carbon (carbon-linked) atom of the fused bicyclic C₄-C₂₀heterocyclyl or fused bicyclic C₁-C₂₀ heteroaryl group R³ group. Fusedbicyclic heterocyclyl and heteroaryl radicals include, but are notlimited to: 1H-indazole, 1H-indole, indolin-2-one,1-(indolin-1-yl)ethanone, 1H-benzo[d][1,2,3]triazole,1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[3,4-d]pyrimidine,1H-benzo[d]imidazole, 1H-benzo[d]imidazol-2(3H)-one,1H-pyrazolo[3,4-c]pyridine, 1H-pyrrolo[2,3-c]pyridine,311-imidazo[4,5-c]pyridine, 7H-pyrrolo[2,3-d]pyrimidine, 7H-purine,1H-pyrazolo[4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine,2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline,isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one,3,4-dihydroquinolin-2(1H)-one, quinazolin-2(1H)-one,quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine,pyrido[3,2-b]pyrazine, benzo[d][1,3]dioxole, and2,3-dihydrobenzo[b][1,4]dioxine.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats or prevents theparticular disease, condition, or disorder, (ii) attenuates,ameliorates, or eliminates one or more symptoms of the particulardisease, condition, or disorder, or (iii) prevents or delays the onsetof one or more symptoms of the particular disease, condition, ordisorder described herein. In the case of cancer, the therapeuticallyeffective amount of the drug may reduce the number of cancer cells;reduce the tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the cancer. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy can be measured, forexample, by assessing the time to disease progression (TTP) and/ordetermining the response rate (RR).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine,dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CASNo. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Il),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the PI3K inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand l or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or l meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, trifluoroacetic acid, maleic acid, succinicacid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalicacid, glycolic acid, salicylic acid, a pyranosidyl acid, such asglucuronic acid or galacturonic acid, an alpha hydroxy acid, such ascitric acid or tartaric acid, an amino acid, such as aspartic acid orglutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid,a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid,or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.The term “hydrate” refers to the complex where the solvent molecule iswater.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and9-fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a “hydroxy-protectinggroup” refers to a substituent of a hydroxy group that blocks orprotects the hydroxy functionality. Suitable protecting groups includeacetyl and silyl. A “carboxy-protecting group” refers to a substituentof the carboxy group that blocks or protects the carboxy functionality.Common carboxy-protecting groups include phenylsulfonylethyl,cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl,2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl,2-(diphenylphosphino)-ethyl, nitroethyl and the like. For a generaldescription of protecting groups and their use, see T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,1991.

The terms “compound of this invention,” and “compounds of the presentinvention” and “compounds of Formula I” include compounds of Formulas Iand stereoisomers, geometric isomers, tautomers, solvates, metabolites,and pharmaceutically acceptable salts and prodrugs thereof.

PYRAZOLO[3,4-D]PYRIMIDINE COMPOUNDS

The present invention provides pyrazolo[3,4-d]pyrimidine compounds, andpharmaceutical formulations thereof, which are potentially useful in thetreatment of diseases, conditions and/or disorders modulated by PI3kinases. More specifically, the present invention provides compounds ofFormula I

and stereoisomers, geometric isomers, tautomers, or pharmaceuticallyacceptable salts thereof, wherein:

R¹ is selected from H, C₁-C₁₂ alkyl, —C(═O)NR¹⁰R¹¹, —NR¹²C(═O)R¹⁰,—NR¹²C(═O)OR¹¹, —NR¹²C(═O)NR¹⁰R¹¹, and C₁-C₂₀ heteroaryl where C₁-C₂₀heteroaryl is optionally substituted with one or more groupsindependently selected from C₁-C₁₂ alkyl, C₁-C₁₂ alkyl-NR¹⁰R¹¹, C₁-C₁₂alkyl-OR¹⁰, C₆-C₂₀ aryl, F, Cl, Br, I, —CN, —CF₃, —CO₂H, —C(═O)NR¹⁰R¹¹,—NO₂, —NR¹⁰R¹¹, —NHCOR¹⁰, —OR¹⁰, —S(O)₂NR¹⁰R¹¹, and —S(O)₂R¹⁰;

R² is C₁-C₁₂ alkyl;

R³ is selected from carbon-linked C₂-C₂₀ heterocyclyl and carbon-linkedC₁-C₂₀ heteroaryl, where carbon-linked C₂-C₂₀ heterocyclyl andcarbon-linked C₁-C₂₀ heteroaryl are optionally substituted with one ormore groups independently selected from C₁-C₁₂ alkyl, C₆-C₂₀ aryl, F,Cl, Br, I, —CH₃, —CN, —CF₃, —CH₂OH, —CO₂H, —CONH₂, —CON(CH₃)₂, —NO₂,—NH₂, —NHCH₃, —NHCOCH₃, —OH, —OCH₃, —SH, —NHC(═O)NHCH₃, and —S(O)₂CH₃;

R⁴ is selected from —NR¹⁰R¹³, —NR¹²C(═O)R¹⁰, —NR¹⁰(C₁-C₁₂ alkyl)NR¹⁰R¹³,—NR¹⁰(C₁-C₁₂ alkyl)OR¹⁰, —NR¹⁰(C₁-C₁₂ alkyl)C(═O)NR¹⁰R¹³, —NR¹⁰(C₁-C₁₂alkylene)-(C₃-C₁₂ carbocyclyl), —NR¹⁰(C₁-C₁₂ alkylene)-(C₂-C₂₀heterocyclyl), —NR¹⁰(C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl), —NR¹⁰(C₁-C₁₂alkylene)-(C₁-C₂₀ heteroaryl), —OR¹⁰, —O(C₁-C₁₂ alkylene)-(C₃-C₁₂carbocyclyl), —O(C₁-C₁₂ alkylene)-(C₂-C₂₀ heterocyclyl), —O(C₁-C₁₂alkylene)-(C₆-C₂₀ aryl), —O(C₁-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl),—(C₁-C₁₂ alkylene)NR¹⁰R¹³, —(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl),—(C₁-C₁₂ alkylene)-(C₂-C₂₀ heterocyclyl), —(C₁-C₁₂ alkylene)-(C₆-C₂₀aryl), —(C₁-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl), —(C₂-C₈alkynylene)NR¹⁰R¹³, —(C₂-C₈ alkynylene)-(C₃-C₁₂ carbocyclyl), —(C₂-C₈alkynylene)-(C₂-C₂₀ heterocyclyl), —(C₂-C₈ alkynylene)-(C₆-C₂₀ aryl),—(C₂-C₈ alkynylene)-(C₁-C₂₀ heteroaryl), —(C₁-C₁₂ alkylene)-(C₆-C₂₀arylene)-(C₂-C₂₀ heterocyclyl), —(C₆-C₂₀ aryl)-(C₁-C₁₂ alkylene)-(C₂-C₂₀heterocyclyl), —C(═O)NR¹⁰R¹¹, C₁-C₁₂ alkyl, C₂-C₈ alkynyl, C₃-C₁₂carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, and C₁-C₂₀ heteroaryl,where alkyl, alkylene, alkynyl, alkynylene, carbocyclyl, heterocyclyl,aryl, and heteroaryl are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂OH, —CN, —CF₃,—CO₂H, —COCH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃,—NHCOCH₃, —NHS(O)₂CH₃, —OH, —OCH₃, —S(O)₂N(CH₃)₂, —SCH₃, —CH₂OCH₃, and—S(O)₂CH₃;

R¹⁰, R¹¹ and R¹² are independently selected from H, C₁-C₁₂ alkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀aryl, and C₁-C₂₀ heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl are optionally substituted with oneor more groups independently selected from F, Cl, Br, I, —CH₂OH,—CH₂C₆H₅, —CN, —CF₃, —CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃,—NHS(O)₂CH₃, —OH, —OCH₃, —OCH₂CH₃, —S(O)₂NH₂, —SCH₃, —S(O)CH₃, —CH₂OCH₃,—CH₃, and —S(O)₂CH₃;

or R¹⁰ and R¹¹ together with the nitrogen atom to which they areattached form a C₂-C₂₀ heterocyclyl ring; and

R¹³ is selected from C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, and C₁-C₂₀ heteroaryl,where alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, —SCH₃, —S(O)CH₃, —OCH₂CH₂—N(CH₃)₂, and—S(O)₂CH₃;

or R¹⁰ and R¹³ together with the nitrogen atom to which they areattached form a C₂-C₂₀ heterocyclyl ring.

Exemplary embodiments include wherein R¹ is H or CH₃.

Exemplary embodiments include wherein R² is CH₃.

Exemplary embodiments include wherein R³ is a monocyclic heteroarylselected from pyridyl, isoxazolyl, imidazolyl, pyrazolyl, pyrrolyl,thiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, oxazolyl, oxadiazolyl,furanyl, thienyl, triazolyl, and tetrazolyl.

Exemplary embodiments include wherein R³ is a fused bicyclic C₄-C₂₀heterocyclyl or C₁-C₂₀ heteroaryl selected from

where the wavy line indicates the site of attachment.

Exemplary embodiments include wherein R³ is selected from:

where the wavy line indicates the site of attachment.

Exemplary embodiments include wherein R³ is a fused bicyclic C₄-C₂₀heterocyclyl or C₁-C₂₀ heteroaryl selected from:

where the wavy line indicates the site of attachment.

Exemplary embodiments include wherein R³ is 1H-indazol-4-yl or1H-indol-4-yl.

Exemplary embodiments include wherein R³ is a monocyclic heteroarylselected from the structures:

where the wavy line indicates the site of attachment.

Exemplary embodiments include wherein R³ is a monocyclic heteroarylselected from the structures:

where the wavy line indicates the site of attachment.

Exemplary embodiments include wherein R³ is a monocyclic heteroarylselected from the structures:

where the wavy line indicates the site of attachment.

Exemplary embodiments include wherein R³ is a C₁-C₂₀ heteroarylsubstituted with one or more groups selected from F, —CF₃, —NH₂, —NHCH₃,—OH, —OCH₃, —C(O)CH₃, —NHC(O)CH₃, —N(C(O)CH₃)₂, —NHC(O)NH₂, —CO₂H, —CHO,—CH₂OH, —C(═O)NHCH₃, —C(═O)NH₂, and —CH₃.

Exemplary embodiments include wherein R⁴ is optionally substitutedpyrimidin-5-yl, optionally substituted C₆-C₂₀ aryl, or optionallysubstituted phenyl.

Exemplary embodiments include wherein R⁴ is —OR¹⁰ where R¹⁰ isoptionally substituted phenyl.

Exemplary embodiments include wherein phenyl is substituted with one ormore groups independently selected from —OCH₃, —SO₂CH₃, —SO₂NH₂,—NHSO₂CH₃, —CH₂OCH₃, —CN, —C(═O)NH₂, —C(═O)NHCH₃, —NHC(═O)CH₃, —CF₃,—OH, —CH₃, and —Cl.

Exemplary embodiments include wherein R⁴ is —OR¹⁰ where R¹⁰ isoptionally substituted pyridyl or optionally substituted C₁-C₁₂ alkyl.

Exemplary embodiments include wherein R⁴ is —O(C₁-C₁₂ alkylene)-(C₂-C₂₀heterocyclyl), —O(C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl), or —O(C₁-C₁₂alkylene)-(C₁-C₂₀ heteroaryl).

Exemplary embodiments include wherein R⁴ is —NR¹⁰R¹³ where R¹⁰ is H,C₁-C₁₂ alkyl, C₂-C₈ alkenyl, or C₂-C₈ alkynyl; and R¹³ is optionallysubstituted phenyl, indazol-6-yl or indazol-4-yl.

Exemplary embodiments include wherein R⁴ is —NR¹⁰(C₁-C₁₂ alkyl)NR¹⁰R¹³,—NR¹⁰(C₁-C₁₂ alkyl)OR¹⁰, or —NR¹⁰(C₁-C₁₂ alkyl)C(═O)NR¹⁰R¹³.

Exemplary embodiments include wherein R⁴ is —NR¹⁰R¹³ where R¹⁰ and R¹³together with the nitrogen atom to which they are attached formmorpholinyl, 4-methylpiperazin-1-yl, 4-methylsulfonylpiperazin-1-yl, or4-(2-pyridyl)piperazin-1-yl.

The Formula I compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention.

In addition, the present invention embraces all geometric and positionalisomers. For example, if a Formula I compound incorporates a double bondor a fused ring, the cis- and trans-forms, as well as mixtures thereof,are embraced within the scope of the invention. Both the singlepositional isomers and mixture of positional isomers are also within thescope of the present invention.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the invention. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The compounds of the present invention may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe invention. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

The present invention also embraces isotopically-labeled compounds ofthe present invention which are identical to those recited herein, butfor the fact that one or more atoms are replaced by an atom having anatomic mass or mass number different from the atomic mass or mass numberusually found in nature. All isotopes of any particular atom or elementas specified are contemplated within the scope of the compounds of theinvention, and their uses. Exemplary isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, ³³P, ³⁵S,¹⁸F, ³⁶Cl, ¹²³I and ¹²⁵I. Certain isotopically-labeled compounds of thepresent invention (e.g., those labeled with ³H and ¹⁴C) are useful incompound and/or substrate tissue distribution assays. Tritiated (³H) andcarbon-14 (¹⁴C) isotopes are useful for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸Fare useful for positron emission tomography (PET) studies to examinesubstrate receptor occupancy. Isotopically labeled compounds of thepresent invention can generally be prepared by following proceduresanalogous to those disclosed in the Schemes and/or in the Examplesherein below, by substituting an isotopically labeled reagent for anon-isotopically labeled reagent.

Preparation of Formula I Compounds

Pyrazolo[3,4-d]pyrimidines compounds of Formula I may be synthesized bysynthetic routes that include processes analogous to those well-known inthe chemical arts, particularly in light of the description containedherein. The starting materials are generally available from commercialsources such as Aldrich Chemicals (Milwaukee, Wis.) or are readilyprepared using methods well known to those skilled in the art (e.g.,prepared by methods generally described in Louis F. Fieser and MaryFieser, Reagents for Organic Synthesis, v. 1-23, Wiley, N.Y. (1967-2006ed.), or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed.Springer-Verlag, Berlin, including supplements (also available via theBeilstein online database).

In certain embodiments, compounds of Formula I may be readily preparedusing well-known procedures to prepare pyrazolo[3,4-d]pyrimidines(Southwick et al (1975) J. of Heterocyclic Chem. 12(6):1199-1205; Chernet al (2004) Bioorganic & Med. Chem. Letters 14(10):2519-2525; Wu et al(2003) Organic Letters 5(20):3587-3590; Quintela et al (2003) Bioorganic& Med. Chem. 11(6):863-868; Braendvang et al (2007) Tetrahedron Letters48(17):3057-3059; Katiyar et al (2006) Synthetic Communications36(20):2963-2973; Holla et al (2006) Bioorganic & Medicinal Chemistry14(6):2040-2047; Kim et al (2003) European Journal of MedicinalChemistry 38 (5), pp 525-532; Quintela et al (2001) European Journal ofMedicinal Chemistry 36 (4), pp 321-332; U.S. Pat. No. 4,001,230; U.S.Pat. No. 4,044,130; U.S. Pat. No. 6,921,763; U.S. Pat. No. 6,660,744;U.S. Pat. No. 7,217,710; US 2006/0128729; US 2007/0155716; WO2005/117909); and other heterocycles, which are described in:Comprehensive Heterocyclic Chemistry II, Editors Katritzky and Rees,Elsevier, 1997, e.g. Volume 3; Liebigs Annalen der Chemie, (9):1910-16,(1985); Helvetica Chimica Acta, 41:1052-60, (1958);Arzneimittel-Forschung, 40(12):1328-31, (1990).

Compounds of Formula I may be prepared singly or as compound librariescomprising at least 2, for example 5 to 1,000 compounds, or 10 to 100compounds. Libraries of compounds of Formula I may be prepared by acombinatorial ‘split and mix’ approach or by multiple parallel synthesesusing either solution phase or solid phase chemistry, by proceduresknown to those skilled in the art. Thus according to a further aspect ofthe invention there is provided a compound library comprising at least 2compounds, or pharmaceutically acceptable salts thereof.

For illustrative purposes, Schemes 1-3 show general methods forpreparing Formula I compounds, as well as key intermediates. For a moredetailed description of the individual reaction steps, see the Examplessection below. Those skilled in the art will appreciate that othersynthetic routes may be used to synthesize the inventive compounds.Although specific starting materials and reagents are depicted in theSchemes and discussed below, other starting materials and reagents canbe easily substituted to provide a variety of derivatives and/orreaction conditions. In addition, many of the compounds prepared by themethods described below can be further modified in light of thisdisclosure using conventional chemistry well known to those skilled inthe art.

In preparing compounds of Formulas I, protection of remote functionality(e.g., primary or secondary amine) of intermediates may be necessary.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York 1991

Scheme 1 shows a general method for preparation of the dihalopyrazolo[3,4-d]pyrimidines intermediate 54 where Hal is independently F,Cl, Br, or I, starting from an ethoxylidene nitrile ester 51.Cyclization of 51a or 51b with an alkyl hydrazine of formula R²NHNH₂,such as methyl hydrazine, yields compounds 52. Treatment of compounds 52with urea at elevated temperatures yields compounds 53. Treatment ofcompounds 53 with a reagent POHal₃, such as phosphoryl chloride yieldscompounds 54.

Scheme 2 shows treatment of compounds 55a or 55b with an alkyl hydrazineR²NHNH₂, such as methyl hydrazine, yielding compounds 56. Hydrolysis ofthe nitrile group, using either sulphuric acid or hydrogen peroxide andsodium hydroxide provides compounds 57. Treatment of compounds 57 withurea at elevated temperatures yields compounds 53. Treatment ofcompounds 53 with a reagent POHal₃, such as phosphoryl chloride and areagent PHal₅, such as phosphorous pentachloride, yields compounds 54.

Scheme 3 shows treatment of a compound 52 with an acylating reagent toform an amide 58 and introduce the R³ substituent. Conversion of theester 58 to primary amide 59 allows for cyclization to 60. Halogenationat the 4 and 6 position gives 4, 6 dichloro pyrazolo[3,4-d]pyrimidine61.

Methods of Separation

In the methods of preparing the compounds of this invention, it may beadvantageous to separate reaction products from one another and/or fromstarting materials. The desired products of each step or series of stepsis separated and/or purified (hereinafter separated) to the desireddegree of homogeneity by the techniques common in the art. Typicallysuch separations involve multiphase extraction, crystallization from asolvent or solvent mixture, distillation, sublimation, orchromatography. Chromatography can involve any number of methodsincluding, for example: reverse-phase and normal phase; size exclusion;ion exchange; high, medium and low pressure liquid chromatographymethods and apparatus; small scale analytical; simulated moving bed(SMB) and preparative thin or thick layer chromatography, as well astechniques of small scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved. For example, boiling point and molecular weightin distillation and sublimation, presence or absence of polar functionalgroups in chromatography, stability of materials in acidic and basicmedia in multiphase extraction, and the like. One skilled in the artwill apply techniques most likely to achieve the desired separation.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine(amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

Biological Evaluation

Determination of the activity of PI3 kinase activity of a compound ofFormula I is possible by a number of direct and indirect detectionmethods. Certain exemplary compounds described herein were assayed fortheir PI3K binding activity (Example 83) and in vitro activity againsttumor cells (Example 84). The range of PI3K binding activities was lessthan 1 nM (nanomolar) to about 10 μM (micromolar). Certain exemplarycompounds of the invention had PI3K binding activity IC₅₀ values lessthan 10 nM. Certain compounds of the invention had tumor cell-basedactivity IC₅₀ values less than 100 nM.

The cytotoxic or cytostatic activity of Formula I exemplary compoundswas measured by: establishing a proliferating mammalian tumor cell linein a cell culture medium, adding a Formula I compound, culturing thecells for a period from about 6 hours to about 5 days; and measuringcell viability (Example 84). Cell-based in vitro assays were used tomeasure viability, i.e. proliferation (IC₅₀), cytotoxicity (EC₅₀), andinduction of apoptosis (caspase activation).

The in vitro potency of Formula I exemplary compounds was measured bythe cell proliferation assay, CellTiter-Glo® Luminescent Cell ViabilityAssay, commercially available from Promega Corp., Madison, Wis. (Example84). This homogeneous assay method is based on the recombinantexpression of Coleoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat.No. 5,674,713; U.S. Pat. No. 5,700,670) and determines the number ofviable cells in culture based on quantitation of the ATP present, anindicator of metabolically active cells (Crouch et al (1993) J. Immunol.Meth. 160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay wasconducted in 96 or 384 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay an be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time.

The anti-proliferative effects of Formula I exemplary compounds weremeasured by the CellTiter-Glo® Assay (Example 84) against several tumorcell lines, including PC3, Detroit 562, and MDAMB361.1. EC₅₀ values wereestablished for the tested compounds. The range of in vitro cell potencyactivities was about 100 nM to about 10 μM.

Certain ADME properties were measured for certain exemplary compounds byassays including: Caco-2 Permeability (Example 85), Hepatocyte Clearance(Example 86), Cytochrome P450 Inhibition (Example 87), Cytochrome P450Induction (Example 88), Plasma Protein Binding (Example 89), and hERGchannel blockage (Example 90).

Exemplary Formula I compounds No. 101-189 in Table 1, were made,characterized, and tested for PI3K activity according to the methods ofthis invention, and have the following structures and correspondingnames (ChemDraw Ultra, Version 9.0.1, CambridgeSoft Corp., CambridgeMass.).

TABLE 1 No. Structure Name 101

N-(3-methoxyphenyl)-1-methyl-6-(1H- pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 102

4-(3,4-dimethoxyphenoxy)-6-(1H-indazol- 4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 103

6-(1H-indazol-4-yl)-1-methyl-4-(4- (methylsulfonyl)phenoxy)-1H-pyrazolo[3,4-d]pyrimidine 104

N-(3-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)methanesulfonamide 105

6-(1H-indazol-4-yl)-4-(3- (methoxymethyl)phenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 106

3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)benzonitrile 107

3-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yl)-N-methylbenzamide 108

6-(1H-indazol-4-yl)-4-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 109

N-(3-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)acetamide 110

6-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4- d]pyrimidine 111

6-(1H-indazol-4-yl)-4-(4-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 112

4-(3,4-dimethoxyphenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 113

6-(1H-indazol-4-yl)-1-methyl-4-(pyridin-3-yloxy)-1H-pyrazolo[3,4-d]pyrimidine 114

6-(1H-indazol-4-yl)-4-(3- methoxyphenoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 115

5-(4-(3-methoxyphenylamino)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-1H- benzo[d]imidazol-2(3H)-one 116

N-(3-methoxyphenyl)-1-methyl-6- (quinolin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 117

N-(3-methoxyphenyl)-1-methyl-6- (quinolin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 118

6-(2-aminopyrimidin-5-yl)-N-(3- methoxyphenyl)-1,3-dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 119

6-(benzo[d][1,3]dioxol-5-yl)-N-(3- methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 120

N-(3-methoxyphenyl)-1-methyl-6-(3-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 121

N-(3-methoxyphenyl)-1-methyl-6-(1H- pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 122

6-(1H-indazol-4-yl)-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4- amine 123

N-(3-methoxyphenyl)-1-methyl-6-(pyridin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4- amine 124

N-(3-methoxyphenyl)-1-methyl-6-(pyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4- amine 125

5-(4-(3-methoxyphenoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)pyridin-2- amine 126

6-(6-aminopyridin-3-yl)-N-(3- methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 127

6-(2-aminopyrimidin-5-yl)-N-(3- methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 128

6-(1H-indazol-4-yl)-1-methyl-N-(3-(methylsulfonyl)benzyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 129

6-(1H-indazol-4-yl)-1-methyl-N-(pyridin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin- 4-amine 130

6-(1H-indazol-4-yl)-N-(2-methoxyethyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4- amine 131

N1-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N1,N2,N2- trimethylethane-1,2-diamine 132

N-(3,4-dimethoxyphenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin- 4-amine 133

6-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-3-yl)ethoxy)-1H-pyrazolo[3,4- d]pyrimidine 134

6-(1H-indazol-4-yl)-1-methyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine 135

6-(1H-indazol-4-yl)-1-methyl-4-(4- (methylsulfonyl)benzyloxy)-1H-pyrazolo[3,4-d]pyrimidine 136

6-(1H-indazol-4-yl)-1-methyl-4-(3-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4- d]pyrimidine 137

4-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzamide 138

4-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzenesulfonamide 139

4-(benzo[d][1,3]dioxol-5-yl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4- d]pyrimidine 140

4-(3,4-dichlorophenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 141

6-(1H-indazol-4-yl)-N-(3-methoxyphenyl)-1,3-dimethyl-1H-pyrazolo[3,4-d]pyrimidin- 4-amine 142

6-(1H-indazol-4-yl)-1-methyl-N-(4-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 143

6-(1H-indazol-4-yl)-1-methyl-N-(2- phenoxyethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 144

6-(1H-indazol-4-yl)-1-methyl-N-(2- (pyridin-3-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 145

6-(1H-indazol-4-yl)-1-methyl-4-(3- (trifluoromethyl)phenethoxy)-1H-pyrazolo[3,4-d]pyrimidine 146

6-(1H-indazol-4-yl)-1-methyl-4- phenethoxy-1H-pyrazolo[3,4-d]pyrimidine147

1-ethyl-6-(1H-indazol-4-yl)-N-(3- methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 148

6-(1H-indazol-4-yl)-1-methyl-4-(4-methylpiperazin-1-yl)-1H-pyrazolo[3,4- d]pyrimidine 149

6-(1H-indazol-4-yl)-1-methyl-4-(4- (methylsulfonyl)piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine 150

4-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yl)-S-dioxothiomorpholine 151

6-(1H-indazol-4-yl)-4-(2-methoxyethoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 152

4-(benzyloxy)-6-(1H-indazol-4-yl)-1- methyl-1H-pyrazolo[3,4-d]pyrimidine153

6-(1H-indazol-4-yl)-4-(3- methoxybenzyloxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 154

6-(1H-indazol-4-yl)-1-methyl-N- (tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 155

6-(1H-indazol-4-yl)-1-methyl-N-(2- morpholinoethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 156

N-(benzo[d][1,3]dioxol-5-yl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 157

N-(4-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)acetamide 158

6-(1H-indazol-4-yl)-1-methyl-4-(3-(pyridin-3-yl)propoxy)-1H-pyrazolo[3,4- d]pyrimidine 159

3-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzamide 160

6-(1H-indazol-4-yl)-1-methyl-N-(3-(4- methylpiperazin-1-yl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 161

N-(4-(2-(dimethylamino)ethoxy)phenyl)-6- (1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 162

6-(1H-indazol-4-yl)-1-methyl-N-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 163

1-(3-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-ylamino)propyl)pyrrolidin-2-one 164

4-(2-(1H-imidazol-1-yl)ethoxy)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4- d]pyrimidine 165

6-(1H-indazol-4-yl)-1-methyl-4-(pyridin-4-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine 166

6-(1H-indazol-4-yl)-1-methyl-4-(3-(pyridin-4-yl)propoxy)-1H-pyrazolo[3,4- d]pyrimidine 167

1-(2-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yloxy)ethyl)pyrrolidin-2-one 168

6-(1H-indazol-4-yl)-1-methyl-N-(3-(pyrrolidin-1-yl)propyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 169

N1-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N3,N3- dimethylpropane-1,3-diamine 170

6-(1H-indazol-4-yl)-1-methyl-N-(3- morpholinopropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 171

6-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-2-yl)ethoxy)-1H-pyrazolo[3,4- d]pyrimidine 172

N-(3-(1H-imidazol-1-yl)propyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 173

6-(1H-indazol-4-yl)-1-methyl-4-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine 174

6-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-4-yl)ethoxy)-1H-pyrazolo[3,4- d]pyrimidine 175

6-(1H-indol-4-yl)-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4- amine 176

1-(4-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yloxy)piperidin-1-yl)ethanone 177

6-(1H-indazol-4-yl)-1-methyl-4-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H- pyrazolo[3,4-d]pyrimidine 178

3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)-1- (piperazin-1-yl)propan-1-one 179

6-(1H-indazol-4-yl)-1-methyl-4-(2-(4-(methylsulfonyl)piperazin-1-yl)ethoxy)- 1H-pyrazolo[3,4-d]pyrimidine 180

6-(1H-indazol-4-yl)-1-methyl-4-(3-((4- (methylsulfonyl)piperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4- d]pyrimidine 181

6-(1H-indazol-4-yl)-1-methyl-4-(4-((4- (methylsulfonyl)piperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4- d]pyrimidine 182

6-(1H-indazol-4-yl)-1-methyl-N-((1- methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 183

6-(1H-indazol-4-yl)-1-methyl-N-(3-(methylsulfonyl)propyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 184

6-(1H-indazol-4-yl)-1-methyl-4-(2-(piperazin-1-yl)ethoxy)-1H-pyrazolo[3,4- d]pyrimidine 185

6-(1H-indazol-4-yl)-1-methyl-4-(piperidin-4-yloxy)-1H-pyrazolo[3,4-d]pyrimidine 186

6-(1H-indazol-4-yl)-1-methyl-N-(3-(methylsulfinyl)propyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 187

6-(1H-indazol-4-yl)-1-methyl-N-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin- 4-amine 188

6-(1H-indazol-4-yl)-1-methyl-N-(3- (methylthio)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 189

4-(2-(6-(1H-indazol-4-yl)-1-methyl-1H- pyrazolo[3,4-d]pyrimidin-4-yloxy)ethyl)morpholine

Administration of Compounds of Formula I

The compounds of the invention may be administered by any routeappropriate to the condition to be treated. Suitable routes includeoral, parenteral (including subcutaneous, intramuscular, intravenous,intra-arterial, intradermal, intrathecal and epidural), transdermal,rectal, nasal, topical (including buccal and sublingual), vaginal,intraperitoneal, intrapulmonary and intranasal. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with for example thecondition of the recipient. Where the compound is administered orally,it may be formulated as a pill, capsule, tablet, etc. with apharmaceutically acceptable carrier or excipient. Where the compound isadministered parenterally, it may be formulated with a pharmaceuticallyacceptable parenteral vehicle and in a unit dosage injectable form, asdetailed below.

A dose to treat human patients may range from about 10 mg to about 1000mg of Formula I compound. A typical dose may be about 100 mg to about300 mg of the compound. A dose may be administered once a day (QID),twice per day (BID), or more frequently, depending on thepharmacokinetic and pharmacodynamic properties, including absorption,distribution, metabolism, and excretion of the particular compound. Inaddition, toxicity factors may influence the dosage and administrationregimen. When administered orally, the pill, capsule, or tablet may beingested daily or less frequently for a specified period of time. Theregimen may be repeated for a number of cycles of therapy.

Methods of Treatment with Formula I Compounds

Compounds of the present invention are useful for treatinghyperproliferative diseases, conditions and/or disorders including, butnot limited to, those characterized by over expression of lipid kinases,e.g. PI3 kinase. Accordingly, another aspect of this invention includesmethods of treating or preventing diseases or conditions that can betreated or prevented by inhibiting lipid kinases, including PI3. In oneembodiment, the method comprises administering to a mammal in needthereof a therapeutically effective amount of a compound of Formula I,or a stereoisomer, geometric isomer, tautomer, or pharmaceuticallyacceptable salt thereof. In one embodiment, a human patient is treatedwith a compound of Formula I and a pharmaceutically acceptable carrier,adjuvant, or vehicle, wherein said compound of Formula I is present inan amount to detectably inhibit PI3 kinase activity.

Cancers which can be treated according to the methods of this inventioninclude, but are not limited to, breast, ovary, cervix, prostate,testis, genitourinary tract, esophagus, larynx, glioblastoma,neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoidcarcinoma, large cell carcinoma, non-small cell lung carcinoma (NSCLC),small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma,pancreas, adenocarcinoma, thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidneycarcinoma, myeloid disorders, lymphoid disorders, hairy cells, buccalcavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine,colon-rectum, large intestine, rectum, brain and central nervous system,Hodgkin's and leukemia.

Another aspect of this invention provides a compound of this inventionfor use in the treatment of the diseases or conditions described hereinin a mammal, for example, a human, suffering from such disease orcondition. Also provided is the use of a compound of this invention inthe preparation of a medicament for the treatment of the diseases andconditions described herein in a warm-blooded animal, such as a mammal,for example a human, suffering from such disorder.

Pharmaceutical Formulations

In order to use a compound of this invention for the therapeutictreatment (including prophylactic treatment) of mammals includinghumans, it is normally formulated in accordance with standardpharmaceutical practice as a pharmaceutical composition. According tothis aspect of the invention there is provided a pharmaceuticalcomposition comprising a compound of this invention in association witha pharmaceutically acceptable diluent or carrier.

A typical formulation is prepared by mixing a compound of the presentinvention and a carrier, diluent or excipient. Suitable carriers,diluents and excipients are well known to those skilled in the art andinclude materials such as carbohydrates, waxes, water soluble and/orswellable polymers, hydrophilic or hydrophobic materials, gelatin, oils,solvents, water and the like. The particular carrier, diluent orexcipient used will depend upon the means and purpose for which thecompound of the present invention is being applied. Solvents aregenerally selected based on solvents recognized by persons skilled inthe art as safe (GRAS) to be administered to a mammal. In general, safesolvents are non-toxic aqueous solvents such as water and othernon-toxic solvents that are soluble or miscible in water. Suitableaqueous solvents include water, ethanol, propylene glycol, polyethyleneglycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof. Theformulations may also include one or more buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents and other known additives to provide an elegant presentation ofthe drug (i.e., a compound of the present invention or pharmaceuticalcomposition thereof) or aid in the manufacturing of the pharmaceuticalproduct (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a compound of Formula I having the desired degree of purity mayoptionally be mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences (1980)16th edition, Osol, A. Ed.), in the form of a lyophilized formulation,milled powder, or an aqueous solution. Formulation may be conducted bymixing at ambient temperature at the appropriate pH, and at the desireddegree of purity, with physiologically acceptable carriers, i.e.,carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8. Formulation in an acetate buffer at pH 5 is asuitable embodiment.

The compound of this invention for use herein is preferably sterile. Inparticular, formulations to be used for in vivo administration must besterile. Such sterilization is readily accomplished by filtrationthrough sterile filtration membranes.

The compound ordinarily can be stored as a solid composition, alyophilized formulation or as an aqueous solution.

The pharmaceutical compositions of the invention will be formulated,dosed and administered in a fashion, i.e., amounts, concentrations,schedules, course, vehicles and route of administration, consistent withgood medical practice. Factors for consideration in this context includethe particular disorder being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat the coagulation factor mediated disorder. Suchamount is preferably below the amount that is toxic to the host orrenders the host significantly more susceptible to bleeding.

As a general proposition, the initial pharmaceutically effective amountof the inhibitor administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg of patient bodyweight per day, with the typical initial range of compound used being0.3 to 15 mg/kg/day.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations of compounds of Formula I may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the administration routesdetailed herein. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Techniques and formulations generally are found inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of a compound of Formula I suitable for oral administrationmay be prepared as discrete units such as pills, capsules, cachets ortablets each containing a predetermined amount of a compound of FormulaI.

Compressed tablets may be prepared by compressing in a suitable machinethe active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and optionally are formulated so as toprovide slow or controlled release of the active ingredient therefrom.

Tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, e.g., gelatincapsules, syrups or elixirs may be prepared for oral use. Formulationsof compounds of Formula I intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium or sodium carbonate, lactose,calcium or sodium phosphate; granulating and disintegrating agents, suchas maize starch, or alginic acid; binding agents, such as starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc. Tablets may be uncoated or may be coated by knowntechniques including microencapsulation to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base.

If desired, the aqueous phase of the cream base may include a polyhydricalcohol, i.e., an alcohol having two or more hydroxyl groups such aspropylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol andpolyethylene glycol (including PEG 400) and mixtures thereof. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active ingredient through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethyl sulfoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier, it desirably comprises a mixture of at least oneemulsifier with a fat or an oil or with both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

Aqueous suspensions of Formula I compounds contain the active materialsin admixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth and gum acacia, and dispersing or wetting agents such asa naturally occurring phosphatide (e.g., lecithin), a condensationproduct of an alkylene oxide with a fatty acid (e.g., polyoxyethylenestearate), a condensation product of ethylene oxide with a long chainaliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such as sucroseor saccharin.

The pharmaceutical compositions of compounds of Formula I may be in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butanediol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

The compounds of Formula I may be employed alone or in combination withother therapeutic agents for the treatment of a disease or disorderdescribed herein, such as a hyperproliferative disorder (e.g., cancer).In certain embodiments, a compound of Formula I is combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound that has anti-hyperproliferativeproperties or that is useful for treating a hyperproliferative disorder(e.g., cancer). The second compound of the pharmaceutical combinationformulation or dosing regimen preferably has complementary activities tothe compound of Formula I such that they do not adversely affect eachother. Such compounds are suitably present in combination in amountsthat are effective for the purpose intended. In one embodiment, acomposition of this invention comprises a compound of Formula I, or astereoisomer, geometric isomer, tautomer, solvate, metabolite, orpharmaceutically acceptable salt or prodrug thereof, in combination witha chemotherapeutic agent such as described herein.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes,separate pills or capsules, or separate infusions. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together.

In a particular embodiment of anti-cancer therapy, a compound of FormulaI, or a stereoisomer, geometric isomer, tautomer, solvate, metabolite,or pharmaceutically acceptable salt or prodrug thereof, may be combinedwith other chemotherapeutic, hormonal or antibody agents such as thosedescribed herein, as well as combined with surgical therapy andradiotherapy. Combination therapies according to the present inventionthus comprise the administration of at least one compound of Formula I,or a stereoisomer, geometric isomer, tautomer, solvate, metabolite, orpharmaceutically acceptable salt or prodrug thereof, and the use of atleast one other cancer treatment method. The amounts of the compound(s)of Formula I and the other pharmaceutically active chemotherapeuticagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect.

Metabolites of Compounds of Formula I

Also falling within the scope of this invention are the in vivometabolic products of Formula I described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,deamidation, esterification, deesterification, enzymatic cleavage, andthe like, of the administered compound. Accordingly, the inventionincludes metabolites of compounds of Formula I, including compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the diseases anddisorders described above is provided. In one embodiment, the kitcomprises a container comprising a compound of Formula I, or astereoisomer, geometric isomer, tautomer, solvate, metabolite, orpharmaceutically acceptable salt or prodrug thereof. The kit may furthercomprise a label or package insert on or associated with the container.The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts. Suitable containers include, for example, bottles, vials,syringes, blister pack, etc. The container may be formed from a varietyof materials such as glass or plastic. The container may hold a compoundof Formula I or a formulation thereof which is effective for treatingthe condition and may have a sterile access port (for example, thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is a compound of Formula I. The label or packageinsert indicates that the composition is used for treating the conditionof choice, such as cancer. In addition, the label or package insert mayindicate that the patient to be treated is one having a disorder such asa hyperproliferative disorder, neurodegeneration, cardiac hypertrophy,pain, migraine or a neurotraumatic disease or event. In one embodiment,the label or package inserts indicates that the composition comprising acompound of Formula I can be used to treat a disorder resulting fromabnormal cell growth. The label or package insert may also indicate thatthe composition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of Formula I and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I, such as tablets or capsules. Sucha kit preferably includes a number of unit dosages. Such kits caninclude a card having the dosages oriented in the order of theirintended use. An example of such a kit is a “blister pack”. Blisterpacks are well known in the packaging industry and are widely used forpackaging pharmaceutical unit dosage forms. If desired, a memory aid canbe provided, for example in the form of numbers, letters, or othermarkings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I contained therein; and optionally (b) asecond container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In certain other embodiments wherein the kit comprises a composition ofFormula I and a second therapeutic agent, the kit may comprise acontainer for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

General Preparative Procedures

General Procedure A Suzuki Coupling:

The Suzuki-type coupling reaction is useful to attach a monocyclicheteroaryl, a fused bicyclic heterocycle, a fused bicyclic heteroaryl,or a phenyl at the 6-position of the pyrimidine ring of a6-chloro-1H-pyrazolo[3,4-d]pyrimidine 21 (see Scheme 4). For example, 21may be combined with 1.5 equivalents of4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1H-indazole 24, anddissolved in 3 equivalents of sodium carbonate as a 1 molar solution inwater and an equal volume of acetonitrile. A catalytic amount, or more,of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. A variety ofboronic acids or boronic esters can be used in place of the indazoleboronic ester indicated. Also alternatively, the nitrogen of theindazole may be protected, for example, N-THP protected compound 41. Insome cases potassium acetate was used in place of sodium carbonate toadjust the pH of the aqueous layer. The reaction is then heated to about140-150° C. under pressure in a microwave reactor such as the BiotageOptimizer (Biotage, Inc.) for 10 to 30 minutes. The contents areextracted with ethyl acetate, or another organic solvent. Afterevaporation of the organic layer the Suzuki coupling products,1,3,4-substituted 6-(1H-indazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidine 22,or 1,3,4-substituted5-(1H-pyrazolo[3,4-d]pyrimidin-6-yl)pyrimidin-2-amine 23, may bepurified on silica or by reverse phase HPLC. Substituents R^(1′),R^(2′), R^(4′) may be R¹, R², R⁴ as defined, or protected forms orprecursors thereof.

Boronic ester (or acid) (1.5 eq) 24 or 25, and a palladium catalyst suchas bis(triphenylphosphine)palladium(II) chloride (0.05 eq) is added to amixture of chloro intermediate (1 eq) 21 in acetonitrile and 1 M ofsodium carbonate aqueous solution (equal volume as acetonitrile). Thereaction mixture is heated to 150° C. in a microwave for 15 min. LC/MSindicates when the reaction is complete. Water is added to the mixture,and the precipitated product is filtered and purified by HPLC to yieldthe product 22 or 23. Substituents R^(1′), R^(2′), R^(4′) may be R¹, R²,R⁴ as defined, or protected forms or precursors thereof.

A variety of palladium catalysts can be used during the Suzuki couplingstep to form compounds, including exemplary embodiments 22 and 23.Suzuki coupling is a palladium mediated cross coupling reaction of anarylhalide, such as 21, with a boronic acid such as 24 or 25. Lowvalent, Pd(II) and Pd(0) catalysts may be used in the Suzuki couplingreaction, including PdCl2(PPH₃)₂, Pd(t-Bu)₃, PdCl₂ dppf CH₂Cl₂,Pd(PPH₃)₄, Pd(OAc)/PPH₃, Cl₂Pd[(Pet₃)]₂, Pd(DIPHOS)₂, Cl₂Pd(Bipy),[PdCl(PH₂PCH₂PPH₂)]₂, Cl₂Pd[P(o-tol)₃]₂, Pd₂(dba)₃/P(o-tol)₃,Pd₂(dba)/P(furyl)₃, Cl₂Pd[P(furyl)₃]₂, Cl₂Pd(PMePH₂)₂,Cl₂Pd[P(4-F-Ph)₃]₂, Cl₂Pd[P(C₆F₆)₃]₂, Cl₂Pd[P(2-COOH-Ph)(Ph)₂]₂,Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂, and encapsulated catalysts Pd EnCat™ 30, PdEnCat™ TPP30, and Pd(II)EnCat™ BINAP30 (US 2004/0254066).

General Procedure B

To a 4,6-dichloro pyrazolo[3,4-d]pyrimidine intermediate 27 in a solventsuch as ethanol is added a primary or secondary amine (R¹⁰R¹³NH, 1.1equiv.) and a non-nucleophilic base such as triethylamine (NEt₃, 1.5 eq,63 μl). Alternatively, acetonitrile may be used as the solvent andpotassium carbonate may be used as the base. The reaction mixture isstirred at room temperature for about 1 hour or overnight, volatilesremoved in vacuo and residue partitioned between DCM and brine. If themixture is insoluble it may be sonicated and the solid product wascollected by filtration. Drying with magnesium sulphate and evaporationof the solvent gives N′-(6-chloro pyrazolo[3,4-d]pyrimidin-4-yl)-aminesubstituted intermediate 28, often as a crystalline solid, or bytrituration. Substituents R^(1′) and R^(2′) may be R¹ and R² as defined,or protected forms or precursors thereof.

General Procedure D

4,6-Dichloropyrazolo[3,4-d]pyrimidine intermediate 27 is added to astirring cold (0° C.) solution of an alcohol R¹⁰OH (1.1 equiv.) andsodium hydride (e.g. 60% wt suspension in mineral oil, 1.4 equiv.) indry THF. The reaction mixture is warmed to room temperature overnight,and then quenched with water. Subsequent DCM/brine extraction andpurification by column chromatography gives 6-chloropyrazolo[3,4-d]pyrimidin-4-yl) alcohol substituted intermediate 30.Substituents R^(1′) and R^(2′) may be R¹ and R² as defined, or protectedforms or precursors thereof.

General Procedure E Suzuki coupling at 4-chloro

4,6-Dichloro pyrazolo[3,4-d]pyrimidine intermediate 27 and an arylboronic acid or pinacol ester (1 eq) are suspended in acetonitrile, andsodium carbonate (3 eq, 80 mg) in solution with water.Bis(triphenylphosphine)palladium(II) chloride (0.05 eq) is added. Thereaction mixture is heated in microwave at about 80° C. for about 5 min.Water is added to the mixture, and the precipitated product is filteredand purified by column chromatography to yield the 4-aryl, 6-chloropyrazolo[3,4-d]pyrimidine product 31. Substituents R^(1′) and R^(2′) maybe R¹ and R² as defined, or protected forms or precursors thereof.

General Procedure F Amide Coupling

A 1,4,6-substituted 1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid 32 istreated with 1.5 eq HATU, 3 eq of an alkylamine (R¹⁰R¹¹NH) and 3 eq ofdiisopropylethylamine (DIPEA) in dimethylformamide (DMF) toapproximately 0.1 M concentration. The reaction is stirred untilcomplete and extracted in ethylacetate with saturated bicarbonatesolution. The organic layer is dried, filtered and concentrated to yieldthe crude intermediate. This intermediate is purified via reverse phaseHPLC to yield product 33 where HPLC R¹ is —C(═O)NR¹⁰R¹¹. SubstituentsR^(2′), R^(3′), R^(4′) may be R², R³, R⁴ as defined, or protected formsor precursors thereof.

EXAMPLES

The chemical reactions described in the Examples may be readily adaptedto prepare a number of other PI3K inhibitors of the invention, andalternative methods for preparing the compounds of this invention aredeemed to be within the scope of this invention. For example, thesynthesis of non-exemplified compounds according to the invention may besuccessfully performed by modifications apparent to those skilled in theart, e.g., by appropriately protecting interfering groups, by utilizingother suitable reagents known in the art other than those described,and/or by making routine modifications of reaction conditions.Alternatively, other reactions disclosed herein or known in the art willbe recognized as having applicability for preparing other compounds ofthe invention.

In the Examples described below, unless otherwise indicated alltemperatures are set forth in degrees Celsius. Reagents were purchasedfrom commercial suppliers such as Sigma Aldrich Chemical Company,Lancaster, TCI or Maybridge, and were used without further purificationunless otherwise indicated. The reactions set forth below were donegenerally under a positive pressure of nitrogen or argon or with adrying tube (unless otherwise stated) in anhydrous solvents, and thereaction flasks were typically fitted with rubber septa for theintroduction of substrates and reagents via syringe. Glassware was ovendried and/or heat dried. Column chromatography was conducted on aBiotage system (Manufacturer: Dyax Corporation) having a silica gelcolumn or on a silica SEP PAK® cartridge (Waters). ¹H NMR spectra wereobtained at 400 MHz in deuterated CDCl₃, d₆-DMSO, CH₃OD or d₆-acetonesolutions (reported in ppm), using chloroform as the reference standard(7.25 ppm). When peak multiplicities are reported, the followingabbreviations are used: s (singlet), d (doublet), t (triplet), m(multiplet), br (broadened), dd (doublet of doublets), dt (doublet oftriplets). Coupling constants, when given, are reported in Hertz (Hz).

Example 1 4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine

Ethoxymethylidenemalonitrile 1 (26.5 g, 0.217 mol) was added portionwise to a mixture of N-methylhydrazine (10 g, 0.217 mol) in ethanol witha speed that the mixture was heated to boiling. The mixture was thenrefluxed for 1 h and cooled down to room temperature. The precipitatedproduct was filtered and washed with ethanol to yield the pure5-amino-1-methyl-1H-pyrazolo 4-carbonitrile 2 (13 g, 50%).

A mixture of 5-amino-1-methyl-1H-pyrazolo 4-carbonitrile 2 (13 g, 0.11mol) in conc. sulfuric acid (30 mL, 0.6 mol) was stirred for 1 h at roomtemperature. The mixture was poured on ice and the resulting mixture wasbasified to pH=8 with 50% ammonia hydroxide aqueous solution in the icebath. The precipitated product was filtered and washed with water togive 5-amino-1-methyl-1H-pyrazolo-4-carboxylic acid amide 3 (13.5 g,91%).

Alternatively, a mixture of 5-amino-1-methyl-1H-pyrazolo 4-carbonitrile2 (3.00 g, 24.59 mmol), 1M aqueous solution of sodium hydroxide (1.1equiv., 27 ml) and ˜30% aqueous solution of hydrogen peroxide (5 equiv.,14 ml) were stirred in methanol (30 ml) at room temperature overnight.The volume of reaction mixture was reduced in vacuo, precipitatefiltered and dried to give 5-amino-1-methyl-1H-pyrazolo-4-carboxylicacid amide 3 (2.99 g, 87%).

A mixture of 5-amino-1-methyl-1H-pyrazolo-4-carboxylic acid amide 3(13.5 g, 0.096 mol) and urea (28.9 g, 0.482 mol) was heated to 180° C.for 2 h. The reaction was cooled down, and the solid was dissolved inhot KOH aq (50%, about 400 mL). The resulting mixture was neutralizedwith acetic acid. The precipitated product was filtered and washed withwater to yield 1-methyl-1,7-dihydro-pyrazolo[3,4-d]-pyrimidine-4,6-dione4 (13.5 g, 84%).

Alternatively, a mixture of 5-amino-1-methyl-1H-pyrazolo-4-carboxylicacid amide 3 (700 mg, 5.00 mmol) and oxalyl chloride (1.2 equiv., 0.5ml) in dry toluene (20 ml) was refluxed overnight. The reaction mixturewas cooled down, precipitate filtered, washed with diethyl ether anddried to give 1-methyl-1,7-dihydro-pyrazolo[3,4-d]pyrimidine-4,6-dione 4(765 mg, 92%).

Phosphorus pentachloride (15 g, 0.072 mol) was added to a mixture of1-methyl-1,7-dihydro-pyrazolo[3,4-d]pyrimidine-4,6-dione 4 (6 g, 0.04mol) in phosphorus oxychloride (10 ml, 0.12 mol). The reaction mixturewas heated to reflux for 18 h. Excess of phosphorus oxychloride wasdistilled off under reduced pressure. The residue was poured on ice. Theresulting mixture was extracted with DCM (3×). The combined organiclayers were washed with water, dried over MgSO₄ and evaporated to yield4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 (4.7 g, 64%).

Example 2 4,6-dichloro-1,3-dimethyl-1H-pyrazolo[3,4-d]pyrimidine 5a

2-(1-Ethoxy-ethylidene)-malononitrile 1a (10 g, 73.45 mmol) was addedportionwise to a mixture of N-methylhydrazine (1 equiv., 3.88 ml) inethanol (50 ml) and the reaction mixture was refluxed for 1 hour, cooledto room temperature, precipitate filtered, washed with ethanol and driedto give 5-amino-1,3-dimethyl-1H-pyrazole-4-carbonitrile 2a as whitecrystalline solid (9.85 g, 68%).

A mixture of 5-amino-1,3-dimethyl-1H-pyrazolo 4-carbonitrile 2a (3.08 g,22.63 mmol), 1M aqueous solution of sodium hydroxide (1.1 equiv., 55 ml)and approximately 30% aqueous solution of hydrogen peroxide (5 eq, 14ml) were stirred in methanol (30 ml) at room temperature for 3 hours.The volume of reaction mixture was reduced in vacuo, precipitatefiltered and dried to give 5-amino-1,3-dimethyl-1H-pyrazolo-4-carboxylicacid amide 3a (3.34 g, 95%).

A mixture of 5-amino-1,3-dimethyl-1H-pyrazolo-4-carboxylic acid amide 3a(3.33 g, 21.61 mmol) and urea (6.5 g, 5 equiv.) was heated to 180° C.for 4 h. The reaction was cooled down, and the solid was dissolved inhot KOH aq (50%, ˜200 mL). The resulting mixture was neutralized with 2Mhydrochloric acid. The precipitated product was filtered and washed withwater to yield1,3-dimethyl-1,7-dihydro-pyrazolo[3,4-d]-pyrimidine-4,6-dione 4a (3.06g).

Phosphorus pentachloride (3.47 g, 3 equiv.) was added to a mixture of1,3-dimethyl-1,7-dihydro-pyrazolo[3,4-d]pyrimidine-4,6-dione 4a (1 g,5.56 mmol) in phosphorus oxychloride (20 ml). The reaction mixture washeated to reflux for 18 h. Excess of phosphorus oxychloride wasdistilled off under reduced pressure. The residue was poured on ice.Extraction into DCM (3×) and purification by column chromatography gave4,6-dichloro-1,3-dimethyl-1H-pyrazolo[3,4-d]pyrimidine 5a (680 mg, 63%)as a beige solid.

Example 3 4,6-Dichloro-1-ethyl-1H-pyrazolo[3,4-d]pyrimidine 5b

Ethoxy-methylidenemalononitrile 1b (1.73 g, 14.17 mmol) was addedportionwise to a mixture of N-ethylhydrazine oxalate (1 equiv., 2.13 ml)and triethylamine (2.1 equiv., 4.17 ml) in ethanol (20 ml) and thereaction mixture was refluxed for 1 hour, cooled to room temperature,precipitate filtered, washed with ethanol and dried to give5-amino-1-ethyl-1H-pyrazole-4-carbonitrile 2b as white crystalline solid(1.02 g, 53%).

A mixture of 5-amino-1-ethyl-1H-pyrazolo 4-carbonitrile 2b (630 mg, 4.65mmol), 1M aqueous solution of sodium hydroxide (1.1 equiv., 5.12 ml) andapproximately 30% aqueous solution of hydrogen peroxide (5 equiv., 2.72ml) were stirred in methanol (30 ml) at room temperature for 3 hours.The volume of reaction mixture was reduced in vacuo, precipitatefiltered and dried to give 5-amino-1-ethyl-1H-pyrazolo-4-carboxylic acidamide 3b (639 g, 78%).

A mixture of 5-amino-1,3-dimethyl-1H-pyrazolo-4-carboxylic acid amide 3b(782 mg, 5.07 mmol) and urea (1.53 g, 5 equiv.) was heated to 180° C.for 2 h. The reaction was cooled down, and the solid was dissolved inhot KOH aq (50%, ˜200 mL). The resulting mixture was neutralized with 2Mhydrochloric acid. The precipitated product was filtered and washed withwater to yield 1-ethyl-1,7-dihydro-pyrazolo[3,4-d]pyrimidine-4,6-dione4b (690 mg).

Phosphorus pentachloride (3.78 g, 3 equiv.) was added to a mixture of1-ethyl-1,7-dihydro-pyrazolo[3,4-d]pyrimidine-4,6-dione 4b (1.09 g, 6.06mmol) in phosphorus oxychloride (15 ml). The reaction mixture was heatedto reflux for 36 h. Excess of phosphorus oxychloride was distilled offunder reduced pressure. The residue was poured on ice. Extraction intoDCM (×3) and purification by column chromatography gave4,6-dichloro-1-ethyl-1H-pyrazolo[3,4-d]pyrimidine 5b (576 mg) as a whitesolid.

Example 46-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6

m-Anisidine (1.1 eq) was added to a solution of4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 (1 eq.) andtriethylamine (NEt₃, 1.5 eq) in ethanol. The mixture was stirred at roomtemperature for 2 hr and then ether was added. The solid was filteredoff and the filtrate concentrated in vacuo to give6-chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6.

Example 5 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole24—Route 1

To a solution of 3-bromo-2-methyl aniline (5.0 g, 26.9 mmol) inchloroform (50 mL) was added potassium acetate (1.05 eq., 28.2 mmol,2.77 g). Acetic anhydride (2.0 eq., 53.7 mmol, 5.07 mL) was added withconcurrent cooling in ice-water. The mixture was then stirred at roomtemperature for 10 minutes after which time a white gelatinous solidformed. 18-Crown-6 (0.2 eq., 5.37 mmol, 1.42 g) was added followed byiso-amyl nitrite (2.2 eq., 59.1 mmol, 7.94 mL) and the mixture washeated under reflux for 18 h. The reaction mixture was allowed to cool,and was partitioned between chloroform (3×100 mL) and saturated aqueoussodium hydrogen carbonate (100 mL). The combined organic extracts werewashed with brine (100 mL), separated and dried (MgSO₄).

The crude product was evaporated onto silica and purified bychromatography eluting with 20% to 40% EtOAc-petrol to give1-(4-bromo-indazol-1-yl)-ethanone A (3.14 g, 49%) as an orange solid,and 4-bromo-1H-indazole B (2.13 g, 40%) as a pale orange solid. A ¹H NMR(400 MHz, CDCl₃) 2.80 (3H, s), 7.41 (1H, t, J=7.8 Hz), 7.50 (1H, d,J=7.8 Hz), 8.15 (1H, s), 8.40 (1H, d, J=7.8 Hz). B: ¹H NMR (400 MHz,CDCl₃) 7.25 (1H, t, J=7.3 Hz), 7.33 (1H, d, J=7.3 Hz), 7.46 (1H, d,J=7.3 Hz), 8.11 (1H, s), 10.20 (1H, br s).

To a solution of the 1-(4-bromo-indazol-1-yl)-ethanone A (3.09 g, 12.9mmol) in MeOH (50 mL) was added 6N aqueous HCl (30 mL) and the mixturewas stirred at room temperature for 7 h. The MeOH was evaporated and themixture partitioned between EtOAc (2×50 mL) and water (50 mL). Thecombined organic layers were washed with brine (50 mL), separated anddried (MgSO₄). The solvent was removed by evaporation under reducedpressure to give 4-bromo-1H-indazole B (2.36 g, 93%).

To a solution of the 4-bromo-1H-indazole B (500 mg, 2.54 mmol) andbis(pinacolato)diboron (1.5 eq., 3.81 mmol) in DMSO (20 mL) was addedpotassium acetate (3.0 eq., 7.61 mmol, 747 mg; dried in drying pistol)and PdCl₂(dppf)₂ (3 mol %, 0.076 mmol, 62 mg). The mixture was degassedwith argon and heated at 80° C. for 40 h. The reaction mixture wasallowed to cool and partitioned between water (50 mL) and ether (3×50mL). The combined organic layers were washed with brine (50 mL),separated and dried (MgSO₄). The crude material was purified bychromatography eluting with 30% to 40% EtOAc-petrol to give aninseparable 3:1 mixture of the4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole 24 (369 mg,60%) and indazole (60 mg, 20%), isolated as a yellow gum whichsolidified upon standing to furnish as an off-white solid. ¹H NMR (400MHz, d₆-DMSO) 1.41 (12H, s), 7.40 (1H, dd, J=8.4 Hz, 6.9 Hz), 7.59 (1H,d, J=8.4 Hz), 7.67 (1H, d, J=6.9 Hz), 10.00 (1H, br s), 8.45 (1H, s),and indazole: 7.40 (1H, t), 7.18 (1H, t, J=7.9 Hz), 7.50 (1H, d, J=9.1Hz), 7.77 (1H, d, J=7.9 Hz), 8.09 (1H, s); impurity at 1.25.

Example 6 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole24—Route 2

To a solution of 2-methyl-3-nitroaniline (2.27 g, 14.91 mmol) in aceticacid (60 mL) was added a solution of sodium nitrite (1.13 g, 1.1 eq.) inwater (5 mL). After 2 h, the deep red solution was poured onto ice/waterand the resulting precipitate collected by filtration to yield4-nitro-1H-indazole C (1.98 g, 81%).

A mixture of 4-nitro-1H-indazole C (760 mg, 4.68 mmol), palladium oncharcoal (10%, cat.) and ethanol (30 mL) was stirred under a balloon ofhydrogen for 4 h. The reaction mixture was then filtered through celite,and the solvent removed in vacuo to yield 1H-indazol-4-ylamine D (631mg, 100%).

An aqueous solution of sodium nitrite (337 mg, 4.89 mmol) in water (2mL) was added dropwise to a suspension of 1H-indazol-4-ylamine D (631mg, 4.74 mmol) in 6M hydrochloric acid (7.2 mL) at below 0° C. Afterstirring for 30 minutes, sodium tetrafluoroborate (724 mg) was added tothe reaction mixture. A viscous solution resulted, which was filteredand washed briefly with water to yield 1H-indazole-4-diazoniumtetrafluoroborate salt E (218 mg, 20%) as a deep red solid.

Dry methanol (4 mL) was purged with argon for 5 minutes. To this wasadded 1H-indazole-4-diazonium tetrafluoroborate salt (218 mg, 0.94mmol), bis-pinacolato diboron (239 mg, 1.0 eq.) and[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chloride (20 mg).The reaction mixture was stirred for 5 h and then filtered throughcelite. The residue was purified using flash chromatography to yield4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole 24 (117mg).

Example 71-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole41 (Route A)

Step A: Preparation of 4-chloro-1H-indazole: To a 250 ml flask with stirbar was added 2-methyl-3-chloroaniline (8.4 ml, 9.95 g, 70.6 mmol),potassium acetate (8.3 g, 84.7 mmol) and chloroform (120 ml). Thismixture was cooled to 0° C. with stirring. To the cooled mixture wasadded acetic anhydride (20.0 ml, 212 mmol) drop wise over 2 minutes. Thereaction mixture was warmed to 25° C. and stirred for 1 hour. At thispoint, the reaction was heated to 60° C. Isoamyl nitrite (18.9 ml, 141mmol) was added and the reaction was stirred overnight at 60° C. Oncecomplete, water (75 ml) and THF (150 ml) were added and the reaction wascooled to 0° C. LiOH (20.7 g, 494 mmol) was added and the reaction wasstirred at 0° C. for 3 hours. Water (200 ml) was added and the productwas extracted with EtOAc (300 ml, 100 ml). The organic layers werecombined, dried with MgSO₄ and concentrated in vacuo to yield4-chloro-1H-indazole 11.07 g (100%) as an orange solid. ¹H NMR (400 MHz,CDCl₃) δ 8.18 (d, J=1 Hz, 1H), 7.33 (d, J=8 Hz 1H), 7.31 (t, J=7 Hz,1H), 7.17 (dd, J=7 Hz, 1 Hz 1H). LCMS (ESI pos) m/e 153 (M+1).

Step B: Preparation of4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole: To a 1 L flask withmechanical stirrer was added 4-chloro-1H-indazole (75.0 g, 0.492 mol),pyridinium p-toluenesulfonate (1.24 g, 4.92 mmol), CH₂Cl₂ (500 ml) and3,4-dihydro-2H-pyran (98.6 ml, 1.08 mol). With stirring, this mixturewas heated to 45° C. for 16 hours. Analysis of reaction mixture showsproduction of both isomers of product. Cooled reaction to 25° C. andadded CH₂Cl₂ (200 ml). Washed the solution with water (300 ml) andsaturated NaHCO₃ (250 ml). Dried the organics with MgSO₄ andconcentrated to dryness. Purified the crude product by dissolving inEtOAc/hexanes (4:6, 1 L) and adding SiO₂ (1.2 L). The mixture wasfiltered and the cake was washed with EtOAc/Hexanes (4:6, 2 L). Theorganics were concentrated in vacuo to yield4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole 110.2 g (95%) as anorange solid. Isomer 1: ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, J=1 Hz, 1H),7.50 (dd, J=9 Hz, 1 Hz 1H), 7.29 (dd, J=9 Hz, 8 Hz 1H), 7.15 (dd, J=8Hz, 1 Hz 1H) 5.71 (dd, J=9 Hz, 3 Hz 1H) 4.02 (m, 1H) 3.55 (m, 1H) 2.51(m, 1H) 2.02 (m, 2H) 1.55 (m, 3H). LCMS (ESI pos) m/e 237 (M+1); Isomer2: ¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J=1 Hz, 1H), 7.62 (dd, J=9 Hz, 1Hz 1H), 7.20 (dd, J=9 Hz, 8 Hz 1H), 7.06 (dd, J=8 Hz, 1 Hz 1H) 5.69 (dd,J=9 Hz, 3 Hz 1H) 4.15 (m, 1H) 3.80 (m, 1H) 2.22 (m, 2H) 2.05 (m, 1H)1.75 (m, 3H). LCMS (ESI pos) m/e 237 (M+1).

Step C: Preparation of1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole41: To a 500 ml flask with stir bar was added4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (10.0 g, 42.2 mmol),DMSO (176 ml), PdCl₂(PPH₃)₂ (6.2 g, 8.86 mmol), tricyclohexylphosphine(0.47 g, 1.69 mmol), bis(pinacolato)diboron (16.1 g, 63.4 mmol) andpotassium acetate (12.4 g, 0.127 mol). With stirring, the mixture washeated to 130° C. for 16 hours. The reaction was cooled to 25° C. andEtOAc (600 ml) was added and washed with water (2×250 ml). The organicswere dried with MgSO₄ and concentrated in vacuo to dryness. The crudeproduct was purified by SiO₂ plug (120 g), eluting with 10%EtOAc/Hexanes (1 L) and 30% EtOAc/Hexanes (1 L). The filtrate wasconcentrated in vacuo to give 13.9 g (100%) of product 41 as a 20%(wt/wt) solution in ethyl acetate. ¹H NMR shows the presence of about20% (wt/wt) bis(pinacolato)diboron. ¹H NMR (400 MHz, CDCl₃) δ 8.37 (s,1H), 7.62 (dd, J=14 Hz, 2 Hz 1H), 7.60 (dd, J=7 Hz, 1 Hz 1H), 7.31 (dd,J=8 Hz, 7 Hz 1H) 5.65 (dd, J=9 Hz, 3 Hz 1H) 4.05 (m, 1H) 3.75 (m, 1H)2.59 (m, 1H) 2.15 (m, 1H) 2.05 (m, 1H) 1.75 (m, 3H) 1.34 (s, 12H). LCMS(ESI pos) m/e 245 (M+1).

Example 81-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1H-indazole41 (Route B)

Step A: Preparation of 4-nitro-1H-indazole: A mixture of2-methyl-3-nitro aniline (200 g, 1.315 moles), acetic acid (8000 ml) wascooled to 15-20° C. and a solution of sodium nitrite (90.6 g, 1.315moles) in water (200 ml) was slowly added over 30 min. After theaddition, the reaction temp. was increased to 25-30° C. and the reactionwas stirred at this temp for 2-3 h. Reaction progress was monitored byTLC and after completion of reaction product was filtered and residuewas washed with acetic acid (1000 ml). Acetic acid was distilled undervacuum (550 mm of Hg) below 80° C. and water (8000 ml) was added, cooledto 25-30° C. and stirred for 30 min. The slurry was filtered and washedwith water (1000 ml). Crude product was dried under heating at 70-80° C.for 2 hours, then was taken in 5% ethyl acetate/n-hexane (100:2000 ml)solution and stirred for 1-1.5 h at ambient temperature. The suspensionwas filtered and washed with 5% ethyl acetate/n-hexane mixture (25:475ml). The product obtained was dried under vacuum at below 80° C. for10-12 h to give 4-nitro-1H-indazole as a brown solid (150 g, 70%): mp:200-203° C.; ¹H NMR (200 MHz, CDCl₃) δ 13.4 (br, 1H), 8.6 (s, 1H),8.2-7.95 (dd, 2H), 7.4 (m, 1H). ESMS m/z 164 (M+1). Purity: 95% (HPLC)

Step B: Preparation of 4-amino-1H-indazole: A mixture of4-nitro-1H-indazole (200 g, 1.22 moles) and 10% palladium on carbon(20.0 g) in EtOH (3000 ml) was hydrogenated at ambient temperature(reaction was exothermic and temperature increased to 50° C.). Aftercompletion of reaction, the catalyst was removed by filtration. Thesolvent was evaporated under vacuum at below 80° C. and cooled to roomtemperature and n-hexane (1000 ml) was added to the residue and stirredfor 30 min. Isolated solid was filtered and washed with n-hexane (200ml). Product was dried under vacuum at 70-80° C. for 10-12 h to give4-amino-1H-indazole as a brown solid (114 g, 70%), m. p.: 136-143° C. ¹HNMR (200 MHz, CDCl₃) δ 12 (br, 1H), 8.0 (s, 1H), 7.1-7.0 (dd, 2H), 6.5(d, 1H), 3.9 (m, 2H). ESMS m/z 134 (M+1). Purity: 90-95% (HPLC)

Step C: Preparation of 4-iodo-1H-indazole: A mixture of4-amino-1H-indazole (50.0 g, 0.375 moles) in water (100 ml) and con.hydrochloric acid (182 ml) was cooled to −10° C. To this a solution ofsodium nitrite (51.7 g, 0.75 moles) in water (75 ml) was added drop wiseat −10° C. in about 30-60 min. (during addition frothing was observed).In another flask a mixture of potassium iodide (311 g, 1.87 moles) inwater (3000 ml) was prepared at room temperature and to this abovecooled diazonium salt at 30-40° C. was added in about 30-40 min. Thereaction was maintained at 30° C. for 1 h and after completion ofreaction, ethyl acetate (500 ml) was added and the reaction mixture wasfiltered through Celite. The layers were separated and the aq. layer wasextracted with ethyl acetate (2×500 ml). The combined organic layerswere washed with 5% hypo solution (2×500 ml), brine (500 ml), dried(Na₂SO₄) and concentrated. Crude product was purified by chromatography(silica gel, hexane, 15-20% ethyl acetate/hexane) to furnish4-iodo-1H-indazole as an orange solid (23.0 g, 25%). mp: 151-177 C: ¹HNMR (200 MHz, CDCl₃) δ 12.4 (br, 1H), 8.0 (s, 1H), 7.6 (dd, 2H), 7.1 (d,1H). ESMS m/z 245 (M+1). Purity: 95-98% (HPLC).

Step D: Preparation of 4-iodo-1-(2-tetrahydropyranyl) indazole: Amixture of 4-amino-1H-indazole (250.0 g, 1.024 moles),3,4-dihydro-2H-pyran (126.0 g, 1.5 moles) and PPTS (2.57 g, 0.01 moles)in CH₂Cl₂ (1250 ml) was heated to 50° C. for 2 h. The reaction wascooled to room temperature and poured into water (625 ml), the layerswere separated, and aqueous layer was extracted with CH₂Cl₂ (250 ml).The combined organic layers were washed with water (625 ml), dried(Na₂SO₄) and concentrated. Crude residue was purified by chromatography(silica gel, hexane, 5-10% ethyl acetate/hexane) to furnish4-iodo-1-(2-tetrahydropyranyl) indazole as an oil (807.0 g, 60%). ¹H NMR(200 MHz, CDCl₃) δ 8.5 (s, 1H), 7.8 (m, 1H), 7.6 (d, 1H), 7.25 (m, 1H),5.7 (dd, 1H), 4.2-3.8 (dd, 1H), 2.2-2.0 (m, 4H) 2.0-1.8 (m, 4H). ESMSm/z 329 (M+1).

Step E: Preparation of1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole41: A mixture of 4-iodo-1-(2-tetrahydropyranyl) indazole (100 g, 0.304moles), bispinacalotodiborane (96.4 g, 0.381 moles), PdCl₂ (dppf) (8.91g, 0.012 moles) and potassium acetate (85.97 g, 0.905 moles) in DMSO(500 ml) were heated to 80° C. for 2-3 h. After completion, reaction wascooled to room temperature and water (1500 ml) was added. Reaction masswas extracted into ethyl acetate (3×200 ml) and combined organic layerswere evaporated, dried (Na₂SO₄) and concentrated. Crude product waspurified by column chromatography (silica gel, hexane, 5-10% ethylacetate/hexane) to obtain 41 as viscous brown oil (70.0 g, 70%). ¹H NMR(CDCl₃) δ 8.5 (s, 1H), 7.8 (m, 1H), 7.6 (d, 1H), 7.25 (m, 1H), 5.7 (dd,1H), 4.2-3.8 (dd, 1H), 2.2-2.0 (m, 4H) 2.0-1.8 (m, 4H) 1.4-1.2 (s, 12H).ESMS m/z 329 (M+1)

Example 9N-(3-methoxyphenyl)-1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine101

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with pyrazole-4-boronic acid pinacol ester using GeneralProcedure A. Addition of water and CH₂Cl₂ to the reaction, followed bysonication and filtration yielded 101. NMR: (CDCl3): 3.87 (s, 3H, CH3),4.07 (s, 3H, CH3), 6.87 (dd, H, ArH, J=2.4, 8.37), 7.13 (d, H, ArH,J=7.85), 7.29 (m, H, ArH), 7.37 (t, H, ArH, J=8.13), 7.43 (s, H, ArH),8.37 (s, 2H, 2×ArH). MS: (ESI+) MH+==322.23

Example 104-(3,4-dimethoxyphenoxy)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine102

General Procedure D and General Procedure A gave 102. NMR (CDCl3): 3.89(3H, s), 4.01 (3H, s), 4.26 (3H, s), 6.93-6.96 (2H, m), 7.03 (1H, d),7.49 (1H, t), 7.63 (1H, d), 7.82 (1H, s), 8.37 (1H, d), 8.82 (1H, s),10.09 (1H, br). MS: MH+ 403

Example 116-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)phenoxy)-1H-pyrazolo[3,4-d]pyrimidine103

General Procedure D and General Procedure A gave 103. NMR (DMSO): 3.38(3H, s), 4.20 (3H, s), 7.45 (1H, t), 7.68 (1H, d), 7.77 (2H, d), 8.04(1H, s), 8.17 (2H, d), 8.20 (1H, d), 8.41 (1H, s), 13.20 (1H, br). MS:MH+ 421.17 (85%)

Example 12N-(3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)methanesulfonamide104

General Procedure E and General Procedure A gave 104. NMR (DMSO-d6):3.11 (3H, s), 4.25 (3H, s), 7.54 (1H, d), 7.61 (1H, t), 7.67 (1H, t),7.81 (1H, d), 8.20 (1H, d), 8.35 (1H, s), 8.57 (1H, d), 8.67 (1H, s),9.11 (1H, s), 10.15 (1H, br), 13.33 (1H, br). MS: MH+ 420.17 (45%)

Example 136-(1H-indazol-4-yl)-4-(3-(methoxymethyl)phenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine105

General Procedure E and General Procedure A gave 105. NMR (CDCl3): 3.52(3H, s), 4.32 (3H, s), 4.68 (2H, s), 7.60-7.71 (4H, m), 8.31 (1H, d),8.35 (1H, s), 8.41 (1H, s), 8.69 (1H, d), 9.30 (1H, s), 10.20 (1H, br).MS: MH+ 371.24 (20%), MH+MeCN 412.24 (100%)

Example 143-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)benzonitrile106

General Procedure E and General Procedure A gave 106. NMR (CDCl3): 4.35(3H, s), 7.63 (1H, m), 7.73 (1H, d), 7.81 (1H, t), 7.93 (1H, d), 8.40(1H, s), 8.61 (1H, dd), 8.68-8.70 (2H, m), 9.26 (1H, s), 10.20 (1H, br).MS: (MH+MeCN) 393.18 (100%)

Example 153-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N-methylbenzamide107

General Procedure E and General Procedure A gave 107. NMR (CDCl3): 3.13(3H, d), 4.33 (3H, s), 6.30 (1H, br), 7.62 (1H, t), 7.70-7.76 (2H, m),8.02 (1H, d), 8.43 (1H, s), 8.51 (1H, d), 8.68 (1H, d), 8.78 (1H, s),9.27 (1H, s), 10.15 (1H, br). MS: MH+ 384.22 (100%)

Example 166-(1H-indazol-4-yl)-4-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine108

General Procedure E and General Procedure A gave 108. NMR (CDCl3): 4.00(3H, s), 4.32 (3H, s), 7.19 (1H, dd), 7.56-7.69 (2H, m), 7.70 (1H, d),7.94-7.96 (2H, m), 8.41 (1H, s), 8.69 (1H, d), 9.31 (1H, s), 10.10 (1H,br). MS: MH+ 357.20 (20%), MH+MeCN 398.21 (100%)

Example 17N-(3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)acetamide109

General Procedure E and General Procedure A gave 109. NMR (CDCl3): 2.30(3H, s), 4.32 (3H, s), 7.39 (1H, s), 7.60-7.64 (2H, m), 7.70 (1H, d),7.80 (1H, d), 8.15 (1H, d), 8.49 (1H, s), 8.59 (1H, s), 8.68 (1H, d),9.30 (1H, s), 10.10 (1H, br). MS: MH+ 384.19 (100%)

Example 186-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine110

General Procedure E and General Procedure A gave 110. NMR (DMSO): 3.36(3H, s), 4.26 (3H, s), 7.60 (1H, t), 7.81 (1H, d), 8.23 (2H, d), 8.58(1H, d), 8.70 (2H, d), 8.78 (1H, s), 9.10 (1H, s). MS: MH+ 405.29 (3%),MH+AcN 446.19 (70%)

Example 196-(1H-indazol-4-yl)-4-(4-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine111

General Procedure E and General Procedure A gave 111. NMR (CDCl3): 3.97(3H, s), 4.30 (3H, s), 7.18 (2H, d), 7.61 (1H, t), 7.69 (1H, d), 8.40(1H, s), 8.41 (2H, d), 8.67 (1H, d), 9.30 (1H, s), 10.15 (1H, br). MS:MH+ 357.22 (85%)

Example 204-(3,4-dimethoxyphenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine112

General Procedure E and General Procedure A gave 112. NMR (CDCl3): 3.95(3H, s), 4.01 (3H, s), 4.21 (3H, s), 7.04 (1H, d), 7.52 (1H, t), 7.60(1H, d), 7.90 (1H, d), 7.96 (1H, s), 8.30 (1H, s), 8.55 (1H, d), 9.20(1H, s), 10.10 (1H, br). MS: MH+ 387.23 (60%)

Example 216-(1H-indazol-4-yl)-1-methyl-4-(pyridin-3-yloxy)-1H-pyrazolo[3,4-d]pyrimidine113

General Procedure D and General Procedure A gave 113. NMR (CDCl3): 4.29(3H, s), 7.44-7.47 (1H, m), 7.51-7.54 (1H, m), 7.57 (1H, d), 7.62 (1H,d), 8.16 (1H, s), 8.25 (1H, d), 8.67-8.70 (2H, m), 8.76 (1H, s), 10.15(1H, br). MS: MH+ 344.23 (15%)

Example 226-(1H-indazol-4-yl)-4-(3-methoxyphenoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine114

A mixture of 3-methoxyphenol (70 μL) and NaH (37 mg) in dry THF (3 mL)under N₂ was stirred for 30 min.4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was added. After 18hr, the reaction mixture was cooled, diluted with water, extracted withEtOAc, dried (Na₂SO₄), filtered and concentrated in vacuo then purifiedby flash chromatography to give a 2:1 mixture of6-chloro-4-(3-methoxy-phenoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine and3-methoxyphenol.

6-Chloro-4-(3-methoxy-phenoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine wasreacted with indazole boronic acid pinacol ester in General Procedure A.Addition of water gave a solid which was purified by preparative HPLC togive 114. NMR: (CDCl3): 3.81 (s, 3H, CH3), 4.18 (s, 3H, CH3), 7.02-7.04(m, 2H, 2×ArH), 7.09 (m, H, ArH), 7.44-7.52 (m, 2H, 2×ArH), 7.70 (d, H,ArH, J=9 Hz), 8.16 (s, H, ArH), 8.22 (d, H, ArH, J=6.9 Hz), 8.34 (s, H,ArH), 13.17 (sbr, H, NH). MS: (ESI+) MH+=373.24

Example 235-(4-(3-methoxyphenylamino)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-1H-benzo[d]imidazol-2(3H)-one115

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with 2-oxo-2,3-dihydro-1H-benzoimidazole-5-boronic acidpinacol ester using General Procedure A. Purification on silica yielded115. NMR: (CDCl3): 3.84 (s, CH3), 4.01 (s, CH3), 6.72 (dd, H, ArH,J=2.1, 8.28), 7.04 (d, H, ArH, J=8.2), 7.35 (t, H, ArH, J=8), 7.47 (d,H, ArH, J=8), 7.72 (s, H, NH), 8.06 (s, H, NH), 8.18 (dd, H, ArH, J=1.4,8.3), 8.25 (s, H, NH), 10.03 (s, H, ArH), 10.81 (s, H, ArH), 10.86 (s,H, ArH). MS: (ESI+) MH+=388.21

Example 24N-(3-methoxyphenyl)-1-methyl-6-(quinolin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine116

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with 5-quinolineboronic acid using General Procedure A.Purification on silica yielded 116. NMR: (CDCl3): 3.80 (s, CH3), 4.13(s, CH3), 6.88 (dd, H, ArH, J=2.3, 8.3), 7.07 (d, H, ArH, J=6.18), 7.22(s, H, NH), 7.35 (t, H, ArH, J=8.1), 7.44-7.51 (m, 3H, 3×ArH), 7.85 (t,H, ArH, J=8.4), 8.25 (d, H, ArH, J=8.4), 8.29 (dd, H, ArH, J=1.1, 8.3),8.98 (dd, H, ArH, J=1.7, 4.1), 9.26 (d, H, ArH, J=8.6). MS: (ESI+)MH+=383.21

Example 25N-(3-methoxyphenyl)-1-methyl-6-(quinolin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine117

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with quinoline-3-boronic acid using General Procedure A.Purification on silica yielded 117. NMR: (CDCl3): 3.90 (s, 3H, CH3),4.17 (s, 3H, CH3), 6.90 (dd, H, ArH, J=2.4, 8.32), 7.20 (d, H, ArH,J=7.92), 7.35 (d, H, ArH, J=11.36), 7.41 (t, H, ArH, J=8.11), 7.51 (s,H, NH), 7.62 (t, H, ArH, J=7.51), 7.79 (t, H, ArH, J=6.970, 8.00 (d, H,ArH, J=8.12), 8.20 (d, H, ArH, J=8.43), 9.30 (d, H, ArH, J=1.8), 10.07(d, H, ArH, J=2.09). MS: (ESI+) MH+=383.23

Example 266-(2-aminopyrimidin-5-yl)-N-(3-methoxyphenyl)-1,3-dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine118

(6-Chloro-1,3-dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(3-methoxy-phenyl)-aminewas reacted with 2-amino-pyrimidine boronic acid pinacol ester usingGeneral Procedure A to give 118. NMR (CDCl3): 2.74 (3H, s), 3.90 (3H,s), 4.04 (3H, s), 5.28 (2H, br), 6.76 (1H, d), 6.99 (1H, br), 7.35 (1H,d), 7.34 (1H, t), 7.55 (1H, s), 9.38 (2H, s). MS: MH+ 363.22 (25%)

Example 276-(benzo[d][1,3]dioxol-5-yl)-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine119

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with 3,4-methylenedioxyphenylboronic acid pinacol esterusing General Procedure A. Purification on silica yielded 119. NMR:(CDCl3): 3.88 (s, 3H, CH3), 4.10 (s, 3H, CH3), 6.07 (s, 2H, CH2), 6.86(dd, H, ArH, J=1.81, 8.3), 6.93 (d, H, ArH, J=8.2), 7.13 (m, 2H, 2×ArH),7.33 (s, H, NH), 7.38 (t, H, ArH, J=8.11), 7.47 (s, H, ArH), 8.06 (d, H,ArH, J=1.54), 8.17 (dd, H, ArH, J=1.63, 8.21). MS: (ESI+) MH+=376.18

Example 28N-(3-methoxyphenyl)-1-methyl-6-(3-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine120

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with 3-methylpyrazole-4-boronic acid pinacol ester usingGeneral Procedure A. Purification on silica yielded 120. NMR: (CDCl3):2.80 (s, 3H, CH3), 3.87 (s, 3H, CH3), 4.06 (s, 3H, CH3), 6.86 (dd, H,ArH, J=1.7, 8.29), 7.14 (dd, H, ArH, J=1.2, 7.89), 7.25 (s, H, ArH),7.36 (t, H, ArH, J=8.11), 7.44 (s, H, ArH), 8.32 (s, H, ArH). MS: (ESI+)MH+=336.24

Example 29N-(3-methoxyphenyl)-1-methyl-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine121

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with 7-azaindole-5-boronic acid pinacol ester usingGeneral Procedure A. Purification on silica yielded 121. NMR: (CDCl3):3.91 (s, 3H, CH3), 4.15 (s, 3H, CH3), 6.66 (m, H, ArH), 6.89 (m, H,ArH), 7.17 (m, H, ArH), 7.24 (s, H, ArH), 7.38-7.42 (m, 3H, 3×ArH), 7.51(s, H, ArH), 9.04 (sbr, H, NH), 9.13 (m, H, ArH), 9.56 (m, H, ArH). MS:(ESI+) MH+=372.25

Example 306-(1H-indazol-4-yl)-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine122

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with indazole boronic acid pinacol ester using GeneralProcedure A. Purification on silica yielded 122. NMR: (CDCl3): 3.88 (s,3H, CH3), 4.19 (s, 3H, CH3), 6.88-6.91 (m, H, ArH), 7.16-7.18 (m, H,ArH), 7.31 (sbr, H, NH), 7.40 (t. H, ArH, J=8.1 Hz), 7.50 (s, H, ArH),7.55 (t. H, ArH, J=4 Hz), 7.65 (d, H, ArH, J=8.3 Hz), 8.45 (d, H, ArH,J=7.49 Hz), 9.17 (s, H, ArH) 10.2 (sbr, H, NH). MS: (ESI+) MH+=372.25

Example 31N-(3-methoxyphenyl)-1-methyl-6-(pyridin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine123

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6 was reacted with pyridine-4-boronic acid pinacol ester using GeneralProcedure A. Purification on silica yielded 123. NMR: (CDCl3): 3.89 (s,3H, CH3), 4.14 (s, 3H, CH3), 6.89-6.92 (m, H, ArH), 7.16 (m, H, ArH),7.25 (s, H, ArH), 7.41 (t, H, ArH, J=8.1 HZ), 7.49 (sbr, H, NH), 8.39(dd, 2H, 2×ArH, J=1.5 Hz, 4.5 Hz), 8.80 (dd, 2H, 2×ArH, J=1.5 Hz, 4.5Hz). MS: (ESI+) MH+=333.24

Example 32N-(3-methoxyphenyl)-1-methyl-6-(pyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine124

6-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine6, from Example 4, was reacted with pyridine-3-boronic acid usingGeneral Procedure A. Purification on silica yielded 124. NMR: (CDCl3):3.88 (s, 3H, CH3), 4.13 (s, 3H, CH3), 6.88-6.90 (m, H, ArH), 7.16 (m, H,ArH), 7.30 (s, H, ArH), 7.38-7.46 (m, 2H, 2×ArH), 7.50 (sbr, H, NH),8.72 (m, H, ArH), 8.79-8.82 (m, H, ArH) 9.76 (m, H, ArH). MS: (ESI+)MH+=333.24

Example 335-(4-(3-methoxyphenoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)pyridin-2-amine125

6-Chloro-4-(3-methoxy-phenoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine (80mg) was coupled to 2-aminopyridine-5-boronic acid pinacol ester viaGeneral Procedure A. The product was purified by reverse phase HPLC toyield 47.3 mg of 125. MS (Q1) 349.2 (M)⁺

Example 346-(6-aminopyridin-3-yl)-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine126

6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine-4-yl)-(3-methoxy-phenyl)-amine(80 mg) was coupled to 2-aminopyridine-5-boronic acid pinacol ester viaGeneral Procedure A and purified by reverse phase HPLC to yield 45 mg of126. MS (Q1) 348.2 (M)⁺.

Example 356-(2-aminopyrimidin-5-yl)-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine127

6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine-4-yl)-(3-methoxyphenyl)-amine(80 mg) was coupled to 2-aminopyrimidine-5-boronic acid pinacol estervia General Procedure A. The product was purified by reverse phase HPLCto yield 8.9 mg of 127. MS (Q1) 349.2 (M)⁺.

Example 366-(1H-indazol-4-yl)-1-methyl-N-(3-(methylsulfonyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine128

Reaction of 3-(methylsulfonyl)benzylamine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(3-methanesulfonyl-benzyl)-amine.

(6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(3-methanesulfonyl-benzyl)-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification on silica yielded 128. NMR (CDCl₃) 3.03 (s,3H, CH3), 4.20 (s, 3H, CH3), 5.13 (d, H, ArH, J=5.95 Hz), 5.84 (sbr, H,NH), 7.51-7.65 (m, 3H, 3×ArH), 7.79 (d, H, ArH, J=7.72 Hz), 7.91 (d, H,ArH, J=7.77 Hz), 7.93 (s, H, ArH), 8.09 (s, H, ArH), 8.39 (d, H, ArH,J=7.28 Hz), 9.04 (s, H, ArH), 10.13 (sbr, H, NH). MS: (ESI+) MH+=434.10

Example 376-(1H-indazol-4-yl)-1-methyl-N-(pyridin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine129

Reaction of 3-aminomethylpyridine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-pyridin-3-ylmethyl-amine.

6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-pyridin-3-ylmethyl-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification on silica yielded 129. NMR (CDCl₃) 4.10 (s,3H, CH3), 4.98 (d, 2H, CH2, J=5.81 Hz), 5.60 (sbr, H, NH), 7.21-7.24 (m,H, ArH), 7.42-7.46 (m, H, ArH), 7.55 (d, H, ArH, J=8.29 Hz), 7.73 (d, H,ArH, J=7.90 Hz), 7.82 (s, H, ArH), 8.33 (d, H, ArH, J=7.27 Hz), 8.51 (m,H, ArH), 8.67 (m, H, ArH), 9.05 (s, H, ArH), 10.05 (sbr, H, NH). MS:(ESI+) MH+=357.14

Example 386-(1H-indazol-4-yl)-N-(2-methoxyethyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine130

Reaction of 2-methoxyethylamine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(2-methoxy-ethyl)-amine.

(6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(2-methoxy-ethyl)-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification on silica yielded 130. NMR: (CDCl3): 3.48 (s,3H, CH3), 3.77 (t, H, ArH, J=5.13 Hz), 4.04 (m, 2H, CH2), 4.18 (s, 3H,CH3), 5.80 (sbr, H, NH), 7.53 (t, H, ArH, J=7.29 Hz), 7.63 (d, H, ArH,J=8.29 HZ), 7.95 (s, H, ArH), 8.43 (d, H, ArH, J=7.24 Hz), 9.17 (s, H,ArH), 10.2 (sbr, H, NH). MS: (ESI+) MH+=324.14

Example 39N1-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N1,N2,N2-trimethylethane-1,2-diamine131

Reaction of N,N,N′-trimethylethylene diamine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gaveN-(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N,N′,N′-trimethyl-ethane-1,2-diamine.

N-(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N,N′,N′-trimethyl-ethane-1,2-diaminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification on silica yielded 131. NMR: (CDCl3): 2.38 (s,6H, 2×CH3), 2.73 (t, 2H, CH2, J=7.17 Hz), 3.54 (s, 311, CH3), 4.08 (m,2H, CH2), 4.17 (s, 3H, CH3), 7.52 (t, H, ArH, J=7.77 Hz), 7.60 (d, H,ArH, J=8.27 Hz), 8.01 (s, H, ArH), 8.42 (d, H, ArH, J=7.23 Hz), 9.19 (s,H, ArH), 10.35 (sbr, H, NH). MS: (ESI+) MH+=351.18

Example 40N-(3,4-dimethoxyphenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine132

Reaction of 4-aminoveratrole with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(3,4-dimethoxy-phenyl)-amine.

(6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(3,4-dimethoxy-phenyl)-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Addition of water gave a solid which was purified bypreparative HPLC to give 132. NMR: (CDCl3): 3.81 (s, 3H, CH3), 3.89 (s,H, ArH), 4.07 (s, 3H, CH3), 6.88 (d, H, ArH, J=8.52 Hz), 7.00 (dd, H,ArH, J=2.41 Hz, 8.46 Hz), 7.15-7.18 (m, 2H, 2×ArH), 7.43-7.47 (m, H,ArH), 7.54-7.56 (m, H, ArH), 8.33 (d, H, ArH, J=7.26 Hz), 9.08 (s, H,ArH), 10.1 (sbr, H, NH). MS: (ESI+) MH+=402.14

Example 416-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-3-yl)ethoxy)-1,1-pyrazolo[3,4-d]pyrimidine133

General Procedure D and General Procedure A gave 133. NMR (CDCl3): 3.30(2H, t), 4.23 (3H, s), 4.99 (2H, t), 7.26-7.31 (1H, m), 7.56 (1H, t),7.66-7.72 (2H, m), 8.05 (1H, s), 8.47 (1H, d), 8.54 (1H, d), 8.67 (1H,s), 9.18 (1H, s), 10.20 (1H, br). MS: MH+ 372.16 (100%)

Example 426-(1H-indazol-4-yl)-1-methyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine134

General Procedure D and General Procedure A gave 134. NMR (DMSO): 4.17(3H, s), 5.89 (2H, s), 7.45-7.48 (1H, m), 7.55 (1H, t), 7.77 (1H, d),8.04 (1H, d), 8.29 (1H, s), 8.45 (1H, d), 8.60 (1H, d), 8.85 (1H, s),9.03 (1H, s), 13.30 (1H, br). MS: MH+ 358.11 (65%)

Example 436-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)benzyloxy)-1H-pyrazolo[3,4-d]pyrimidine135

General Procedure D and General Procedure A gave 135. NMR (CDCL3): 3.08(3H, s), 4.26 (3H, s), 5.92 (2H, s), 7.57 (1H, t), 7.69 (1H, d), 7.79(2H, d), 8.02 (2H, d), 8.12 (1H, s), 8.45 (1H, d), 9.12 (1H, s), 10.20(1H, br). MS: MH+ 435.08 (100%)

Example 446-(1H-indazol-4-yl)-1-methyl-4-(3-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine136

General Procedure E and General Procedure A gave 136. NMR (CDCl3): 3.21(3H, s), 4.35 (3H, s), 7.63 (1H, t), 7.73 (1H, d), 7.91 (1H, t), 8.22(1H, d), 8.43 (1H, s), 8.67-8.71 (2H, m), 8.96 (1H, s), 9.26 (1H, s),10.20 (1H, br). MS: MH+ 404.17 (5%), MH+AcN 446.10 (100%)

Example 454-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzamide137

General Procedure E and General Procedure A gave 137. NMR (CDCl3): 3.10(3H, br), 3.21 (3H, br), 4.33 (3H, s), 7.62 (1H, t), 7.71 (2H, d), 8.40(1H, s), 8.42 (2H, d), 8.68 (1H, d), 9.29 (1H, s), 10.20 (1H, br). MS:MH+ 398.18 (5%), MH+AcN 439.19 (100%)

Example 464-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzenesulfonamide138

General Procedure E and General Procedure A gave 138. NMR (CDCl3): 2.85(6H, s), 4.35 (3H, s), 7.63 (1H, t), 7.73 (1H, d), 8.08 (2H, d), 8.41(1H, s), 8.54 (2H, d), 8.69 (1H, d), 9.28 (1H, s), 10.20 (1H, br). MS:MH+ 434.18 (5%), MH+AcN 475.11 (100%)

Example 474-(benzo[d][1,3]dioxol-5-yl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine139

General Procedure E and General Procedure A gave 139. NMR (CDCl3): 4.30(3H, s), 6.14 (2H, s), 7.08 (1H, d), 7.60 (1H, t), 7.69 (1H, d), 7.92(1H, s), 7.95 (1H, d), 8.37 (1H, s), 8.66 (1H, d), 9.28 (1H, s), 10.20(1H, br). MS: MH+ 371.26 (30%)

Example 484-(3,4-dichlorophenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine140

General Procedure E and General Procedure A gave 140. NMR (CDCl3): 4.33(3H, s), 7.63 (1H, t), 7.71-7.76 (2H, m), 8.22 (1H, dd), 8.38 (1H, s),8.49 (1H, s), 8.67 (1H, d), 9.25 (1H, s), 10.20 (1H, br). MS: MH+AcN436.11 (100%)

Example 496-(1H-indazol-4-yl)-N-(3-methoxyphenyl)-1,3-dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine141

(6-Chloro-1,3-dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(3-methoxyphenyl)-aminewas reacted with indazole boronic acid pinacol ester using GeneralProcedure A to give 141. NMR (CDCl3): 2.78 (3H, s), 3.89 (3H, s), 4.14(3H, s), 6.79 (1H, dd), 7.02 (1H, br. s), 7.25 (1H, m), 7.37 (1H, t),7.54 (1H, t), 7.63-7.68 (2H, m), 8.45 (1H, d), 9.10 (1H, s), 10.11 (1H,br). MS: MH+ 386.20 (100%)

Example 506-(1H-indazol-4-yl)-1-methyl-N-(4-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine142

To 4-(methylsulfonyl)aniline (500 mg, 2.93 mmol) in anhydrous DCM (5 ml)was added di-tert-butyldicarbonate (1.2 equiv., 768 mg) and DMAP (cat.).The reaction mixture was stirred at room temperature overnight.DCM/brine extraction and column chromatography gave(4-methanesulfonyl-phenyl)carbamic acid tert-butyl ester as a whitesolid (280 mg).

To (4-methanesulfonyl-phenyl)carbamic acid tert-butyl ester (280 mg) indry THF (3 ml) was added sodium hydride (60% wt suspension in mineraloil, 1.4 equiv., 33.3 mg) at 0° C. After the effervescence ceased, added4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 (113 mg) and thereaction mixture was allowed to warm up to room temperature overnight.DCM/brine extraction gave crude(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(4-methanesulfonyl-phenyl)carbamicacid tert-butyl ester, which was reacted with indazole boronic acidpinacol ester in General Procedure A to give 142 as a beige solid (118mg). NMR (d6-DMSO): 3.24 (3H, s), 4.15 (3H, s), 7.56 (1H, t), 7.74 (1H,d), 8.01 (2H, d), 8.28 (2H, d), 8.32 (1H, d), 8.41 (1H, s), 8.99 (1H,s), 10.54 (1H, br), 13.25 (1H, br). MS: MH+ 420.13

Example 516-(1H-indazol-4-yl)-1-methyl-N-(2-phenoxyethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine143

Reaction of 2-phenoxyethylamine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(2-phenoxy-ethyl)-amine.

(6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(2-phenoxy-ethyl)-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification by preparative HPLC yielded 143. NMR (CDCl₃)4.19 (s, 3H, CH3), 4.27 (m, 2H, CH2), 4.36 (m, 2H, CH3), 5.83 (sbr, H,NH), 7.00 (m, 3H, 3×ArH), 7.32 (m, 2H, 2×ArH), 7.55 (m, H, ArH), 7.64(m, H, ArH), 7.96 (s, H, ArH), 8.45 (d, H, ArH, J=7.23 Hz0, 9.18 (s, H,ArH), 10.16 (sbr, H, NH). MS: (ESI+) MEI+=386.19

Example 526-(1H-indazol-4-yl)-1-methyl-N-(2-(pyridin-3-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine144

Reaction of 3-(2-aminoethyl)pyridine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(2-pyridin-3-yl-ethyl)-amine.

(6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(2-pyridin-3-yl-ethyl)-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification by preparative HPLC yielded 144. NMR (CDCl₃)3.08 (t, H, CH2, J=7.06 Hz), 4.01 (m, 2H, CH2), 4.09 (s, 3H, CH3), 5.4(sbr, H, NH), 7.20 (m, H, ArH), 7.46 (m, H, ArH), 7.55 (m, 2H, 2×ArH),7.84 (s, H, ArH), 8.35 (d, H, ArH, J=7.21 Hz), 8.45 (m, H, ArH), 8.50(s, H, ArH), 10.13 (sbr, H, NH). MS: (ESI+) MH+=371.15

Example 536-(1H-indazol-4-yl)-1-methyl-4-(3-(trifluoromethyl)phenethoxy)-1H-pyrazolo[3,4-d]pyrimidine145

General Procedure D and General Procedure A gave 145. NMR (CDCl3): 3.35(2H, t), 4.23 (3H, s), 5.00 (2H, t), 7.45-7.50 (1H, m), 7.54-7.59 (3H,m), 7.67-7.70 (2H, m), 8.04 (1H, s), 8.47 (1H, d), 9.18 (1H, s), 10.15(1H, br). MS: MH+ 439.12 (35%), MH+AcN 480.19 (100%)

Example 546-(1H-indazol-4-yl)-1-methyl-4-phenethoxy-1H-pyrazolo[3,4-d]pyrimidine146

General Procedure D and General Procedure A gave 146. NMR (CDCl3): 3.30(2H, t), 4.23 (3H, s), 4.98 (2H, t), 7.27-7.31 (1H, m), 7.35-7.40 (4H,m), 7.55 (1H, t), 7.67 (1H, d), 8.06 (1H, s), 8.49 (1H, d), 9.19 (1H,s), 10.10 (1H, br). MS: MH+ 371.13 (50%), MH+AcN 412.12 (100%)

Example 551-ethyl-6-(1H-indazol-4-yl)-N-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine147

4,6-Dichloro-1-ethyl-1H-pyrazolo[3,4-d]pyrimidine 5b was reacted withm-anisidine using General Procedure B to give(6-chloro-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(3-methoxy-phenyl)-amine,which was reacted with indazole boronic acid pinacol ester using GeneralProcedure A to give 147. NMR (CDCl3): 1.62 (3H, t), 3.88 (3H, s), 4.62(2H, q), 6.89 (1H, dd), 7.17 (1H, d), 7.32 (1H, s), 7.40 (1H, t), 7.51(1H, s), 7.55 (1H, t), 7.64 (1H, d), 8.45 (1H, d), 9.16 (1H, s), 10.20(1H, br). MS: MH+ 386.19 (100%)

Example 566-(1H-indazol-4-yl)-1-methyl-4-(4-methylpiperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine148

Reaction of N-methylpiperazine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave6-chloro-1-methyl-4-(4-methyl-piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine.

6-Chloro-1-methyl-4-(4-methyl-piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidinewas reacted with indazole boronic acid pinacol ester using GeneralProcedure A. Purification on silica yielded 148. NMR (CDCl3): 2.41 (3H,s), 2.60-2.66 (4H, m), 4.15-4.19 (7H, m), 7.50-7.53 (1H, m), 7.61 (1H,d), 8.00 (1H, s), 8.41 (1H, d), 9.13 (1H, s). MS: (ESI+) MH+=349

Example 576-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine149

Reaction of 1-methanesulfonyl-piperazine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave6-chloro-4-(4-methanesulfonyl-piperazin-1-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine.

6-Chloro-4-(4-methanesulfonyl-piperazin-1-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine wasreacted with indazole boronic acid pinacol ester using General ProcedureA. Purification on silica yielded 149. NMR: (DMSO) 2.98 (3H, s),3.35-3.40 (4H, m), 4.11 (3H, s), 4.18-4.22 (4H, m), 7.48-7.51 (1H, m),7.70 (1H, d), 8.30 (1H, d), 8.42 (1H, s), 8.95 (1H, s). 13.20 (1H, s).MS: (ESI+) MH+=413

Example 584-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-S-dioxothiomorpholine150

Reaction of thiomorpholine-1,1-dioxide with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 by GeneralProcedure B gave6-chloro-4-(1,1-dioxo-thiomorpholin-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine.

6-Chloro-4-(1,1-dioxo-thiomorpholin-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidinewas reacted with indazole boronic acid pinacol ester using GeneralProcedure A. Purification on silica yielded 150. NMR: (DMSO) 3.45-3.50(4H, m), 4.11 (3H, s), 4.45-4.50 (4H, m), 7.48-7.51 (1H, m), 7.70 (1H,d), 8.30 (1H, d), 8.38 (1H, s), 8.95 (1H, s). 13.20 (1H, s). MS: (ESI+)MH+=425

Example 596-(1H-indazol-4-yl)-4-(2-methoxyethoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine151

General Procedure D and General Procedure A gave 151. NMR (CDCl3): 3.52(3H, s), 3.94 (2H, t), 4.23 (3H, s), 4.94 (2H, t), 7.56 (1H, t), 7.67(1H, d), 8.12 (1H, s), 8.49 (1H, d), 9.18 (1H, s), 10.15 (1H, br). MS:MH+ 325.12 (20%), 366.15 (100%)

Example 604-(benzyloxy)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine152

General Procedure D and General Procedure A gave 152. NMR (CDCl3): 4.24(3H, s), 5.84 (2H, s), 7.38-7.47 (3H, m), 7.56-7.60 (3H, m), 7.68 (1H,d), 8.09 (1H, s), 8.52 (1H, d), 9.18 (1H, s), 10.15 (1H, br). MS: MH+357.12 (35%), MH+AcN 398.15 (100%)

Example 616-(1H-indazol-4-yl)-4-(3-methoxybenzyloxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine153

General Procedure D and General Procedure A gave 153. NMR (CDCl3): 3.84(3H, s), 4.24 (3H, s), 5.81 (2H, s), 6.93 (1H, dd), 7.15-7.19 (2H, m),7.36 (1H, t), 7.57 (1H, t), 7.67 (1H, d), 8.10 (1H, s), 8.52 (1H, d),9.19 (1H, s), 10.15 (1H, br). MS: MH+ 387.14 (100%)

Example 626-(1H-indazol-4-yl)-1-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine154

Reaction of 4-aminotetrahydropyran with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B to(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(tetrahydropyran-4-yl)-amine.

(6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(tetrahydro-pyran-4-yl)-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification on silica yielded 154. NMR (CDCl3) 1.70-1.80(m, 2H, CH2), 2.24-2.27 (m, 2H, CH2), 3.67-3.73 (m, 2H, CH2), 4.11-4.14(m, 2H, CH2), 4.18 (s, 3H, CH3), 4.55 (m, H, CH), 5.32 (sbr, H, NH),7.55 (t, H, ArH, J=7.76 Hz), 7.63 (d, H, ArH, J=8.27 Hz), 7.92 (s, H,ArH), 8.40 (d, H, ArH, J=7.23 Hz), 9.16 (s, H, ArH), 10.22 (sbr, H, NH).MS: (ESI+) MH+=350.16

Example 636-(1H-indazol-4-yl)-1-methyl-N-(2-morpholinoethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine155

Reaction of 4-(2-aminoethyl)morpholine with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B to(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(2-morpholin-4-yl-ethyl)-amine.

(6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-(2-morpholin-4-yl-ethyl)-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification on silica yielded 155. NMR (CDCl₃) 2.50 (m,4H, 2×CH2), 2.70 (t, 2H, CH2, J=5.95 Hz), 3.71 (m, 4H, 2×CH2), 3.80 (m,2H, CH2), 4.08 (s, 3H, CH3), 3.05 (sbr, H, NH), 7.44 (t, H, ArH, J=7.32Hz), 7.54 (d, H, ArH, J=XXHz), 7.87 (s, H, ArH), 8.35 (m, H, ArH), 9.08(s, H, NH). MS: (ESI+) MH+=379.17

Example 64N-(benzo[d][1,3]dioxol-5-yl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine156

Reaction of 3,4-(methylenedioxy)aniline with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B tobenzo[1,3]dioxol-5-yl-(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amine.

Benzo[1,3]dioxol-5-yl-(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-aminewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Addition of water gave a solid which was purified bypreparative HPLC and trituration to give 156. NMR: (CDCl3): 3.52 (s, 3H,CH3), 4.17 (s, 3H, CH3), 6.10 (s, 2H, CH2), 6.92 (d, H, ArH, J=8.16 Hz),7.00 (d, H, ArH, J=8.16 Hz), 7.10 (s, H, ArH), 7.16 (sbr, H, NH), 7.23(s, H, ArH), 7.55 (t, H, ArH, J=8.17 Hz), 7.64 (d, H, ArH, J=8.31 Hz0,8.41 (d, H, ArH, J=7.26 Hz), 9.16 (s, H, ArH), 10.20 (sbr, H, NH). MS:(ESI+) MH+=386.04

Example 65N-(4-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)acetamide157

Reaction of 4-aminoacetanilide with4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B toN-[4-(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)-phenyl]-acetamide.

N-[4-(6-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)-phenyl]-acetamidewas reacted with indazole boronic acid pinacol ester in GeneralProcedure A. Purification on silica and trituration yielded 157. NMR:(CDCl3): 2.08 (s, 3H, CH3), 4.17 (s, 3H, CH3), 7.51 (t, H, ArH, J=7.77Hz), 7.69 (m, 3H, 3×ArH), 7.78 (m, 2H, 2×ArH), 8.28 (d, H, ArH, J=7.26Hz), 8.17 (sbr, H, NH), 8.93 (s, H, ArH), 9.98 (s, H, ArH), 10.07 (s, H,ArH), 13.18 (s, H, ArH). MS: (ESI+) MH+=399.13

Example 666-(1H-indazol-4-yl)-1-methyl-4-(3-(pyridin-3-yl)propoxy)-1H-pyrazolo[3,4-d]pyrimidine158

General Procedure D and General Procedure A gave 158. NMR (CDCl3): 2.23(2H, quintet), 2.84 (2H, t), 4.14 (3H, s), 4.72 (2H, t), 7.14-7.26 (1H,m), 7.45 (1H, t), 7.51 (1H, d), 7.58 (1H, d), 7.97 (1H, s), 8.35 (1H,d), 8.40 (1H, d), 8.48 (1H, s), 9.07 (1H, s), 10.20 (1H, br). MS: MH+386.11 (100%)

Example 673-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzamide159

General Procedure E and General Procedure A gave 159. NMR (CDCl3): 3.14(3H, br), 3.22 (3H, br), 4.33 (3H, s), 7.61 (1H, t), 7.69-7.74 (3H, m),8.40-8.45 (3H, m), 8.68 (1H, d), 9.27 (1H, s), 10.20 (1H, br). MS: MH+398.16 (100%).

Example 686-(1H-indazol-4-yl)-1-methyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine160

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 160.

Example 69N-(4-(2-(dimethylamino)ethoxy)phenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine161

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 161.

Example 706-(1H-indazol-4-yl)-1-methyl-N-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine162

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 162.

Example 711-(3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)propyl)pyrrolidin-2-one163

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 163.

Example 724-(2-(1H-imidazol-1-yl)ethoxy)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine164

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 164.

Example 736-(1H-indazol-4-yl)-1-methyl-4-(pyridin-4-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine165

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 165.

Example 746-(1H-indazol-4-yl)-1-methyl-4-(3-(pyridin-4-yl)propoxy)-1H-pyrazolo[3,4-d]pyrimidine166

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 166.

Example 751-(2-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)ethyl)pyrrolidin-2-one167

4,6-Dichloro-1-methyl-1,1-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 167.

Example 766-(1H-indazol-4-yl)-1-methyl-N-(3-(pyrrolidin-1-yl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine168

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 168.

Example 77N1-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N3,N3-dimethylpropane-1,3-diamine169

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 169.

Example 786-(1H-indazol-4-yl)-1-methyl-N-(3-morpholinopropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine170

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 170.

Example 796-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-2-yl)ethoxy)-1H-pyrazolo[3,4-d]pyrimidine171

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure D and General Procedure A to give 171.

Example 80N-(3-(1H-imidazol-1-yl)propyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine172

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 172.

Example 816-(1H-indazol-4-yl)-1-methyl-4-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine173

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure B and General Procedure A to give 173.

Example 826-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-4-yl)ethoxy)-1H-pyrazolo[3,4-d]pyrimidine174

4,6-Dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine 5 was converted byGeneral Procedure D and General Procedure A to give 174.

Example 83 p110α (Alpha) PI3K Binding Assay

Binding Assays: Initial polarization experiments were performed on anAnalyst HT 96-384 (Molecular Devices Corp, Sunnyvale, Calif.). Samplesfor fluorescence polarization affinity measurements were prepared byaddition of 1:3 serial dilutions of p110 alpha PI3K (Upstate CellSignaling Solutions, Charlottesville, Va.) starting at a finalconcentration of 20 ug/mL in polarization buffer (10 mM Tris pH 7.5, 50mM NaCl, 4 mM MgCl₂, 0.05% Chaps, and 1 mM DTT) to 10 mM PIP₂(Echelon-Inc., Salt Lake City, Utah.) final concentration. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah.) 100 nM and 5 nM final concentrationsrespectively. Read with standard cut-off filters for the rhodaminefluorophore (λex=530 nm; λem=590 nm) in 384-well black low volumeProxiplates (PerkinElmer, Wellesley, Mass.) Fluorescence polarizationvalues were plotted as a function of the protein concentration, and theEC₅₀ values were obtained by fitting the data to a 4-parameter equationusing KaleidaGraph software (Synergy software, Reading, Pa.). Thisexperiment also establishes the appropriate protein concentration to usein subsequent competition experiments with inhibitors.

Inhibitor IC₅₀ values were determined by addition of the 0.04 mg/mL p110alpha PI3K (final concentration) combined with PIP₂ (10 mM finalconcentration) to wells containing 1:3 serial dilutions of theantagonists in a final concentration of 25 mM ATP (Cell SignalingTechnology, Inc., Danvers, Mass.) in the polarization buffer. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah.) 100 nM and 5 nM final concentrationsrespectively. Read with standard cut-off filters for the rhodaminefluorophore (λex=530 nm; λem=590 nm) in 384-well black low volume proxiplates (PerkinElmer, Wellesley, Mass.) Fluorescence polarization valueswere plotted as a function of the antagonist concentration, and the IC₅₀values were obtained by fitting the data to a 4-parameter equation inAssay Explorer software (MDL, San Ramon, Calif.).

Alternatively, inhibition of PI3K was determined in a radiometric assayusing purified, recombinant enzyme and ATP at a concentration of 1 uM.The Formula I compound was serially diluted in 100% DMSO. The kinasereaction was incubated for 1 h at room temperature, and the reaction wasterminated by the addition of PBS. IC₅₀ values were subsequentlydetermined using sigmoidal dose-response curve fit (variable slope).

Example 84 In Vitro Cell Proliferation Assay

Efficacy of Formula I compounds were measured by a cell proliferationassay employing the following protocol (Promega Corp. Technical BulletinTB288; Mendoza et al (2002) Cancer Res. 62:5485-5488):

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells (PC3,Detroit562, or MDAMB361.1) in medium was deposited in each well of a384-well, opaque walled plate.

2. Control wells were prepared containing medium and without cells.

3. The compound was added to the experimental wells and incubated for3-5 days.

4. The plates were equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well was added.

6. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence was recorded and reported in graphs as RLU=relativeluminescence units.

Alternatively, cells were seeded at optimal density in a 96 well plateand incubated for 4 days in the presence of test compound. Alamar Blue™was subsequently added to the assay medium, and cells were incubated for6 h before reading at 544 nm excitation, 590 nm emission. EC₅₀ valueswere calculated using a sigmoidal dose response curve fit.

Example 85 Caco-2 Permeability

Caco-2 cells are seeded onto Millipore Multiscreen plates at 1×10⁵cells/cm², and cultured for 20 days. Assessment of compound permeabilityis subsequently conducted. The compounds are applied to the apicalsurface (A) of cell monolayers and compound permeation into thebasolateral (B) compartment was measured. This is performed in thereverse direction (B−A) to investigate active transport. A permeabilitycoefficient value, P_(app), for each compound, a measure of the rate ofpermeation of the compound across the membrane, is calculated. Compoundsare grouped into low (P_(app)</=1.0×10⁶ cm/s) or high (P_(app)>/=1.0×10⁶cm/s) absorption potential based on comparison with control compoundswith established human absorption.

For assessment of a compound's ability to undergo active efflux, theratio of basolateral (B) to apical (A) transport compared with A to Bwas determined. Values of B−A/A−B>/=1.0 indicate the occurrence ofactive cellular efflux.

Example 86 Hepatocyte Clearance

Suspensions of cryopreserved human hepatocytes are used. Incubations areperformed at compound concentration of 1 mM or 3 μM at a cell density of0.5×10⁶ viable cells/mL. The final DMSO concentration in the incubationis about 0.25%. Control incubations are also performed in the absence ofcells to reveal any non-enzymatic degradation. Duplicate samples (5 μL)are removed from the incubation mixture at 0, 5, 10, 20, 40 and 60minutes (control sample at 60 minutes only) and added to MeOH-containinginternal standard (100 μL)—to terminate the reaction. Tolbutamide,7-hydroxycoumarin, and testosterone may be used as control compounds.Samples are centrifuged and the supernatants at each time point pooledfor analysis by LC-MSMS. From a plot of ln peak area ratio (parentcompound peak area/internal standard peak area) against time, intrinsicclearance (CL_(int)) is calculated as follows: CL_(int) (μl/min/millioncells)=V×k, where k is the elimination rate constant, obtained from thegradient of ln concentration plotted against time; V is a volume termderived from the incubation volume and is expressed as uL 10⁶ cells⁻¹.

Example 87 Cytochrome P450 Inhibition

Formula I compounds may be screened against CYP450 targets (1A2, 2C9,2C19, 2D6, 3A4) at about 10 concentrations in duplicate, with a topconcentration of about 100 uM. Standard inhibitors (furafylline,sulfaphenazole, tranylcypromine, quinidine, ketoconazole) may be used ascontrols. Plates may be read using a BMG LabTechnologies PolarStar influorescence mode.

Example 88 Cytochrome P450 Induction

Freshly isolated human hepatocytes from a single donor may be culturedfor about 48 h prior to addition of Formula I compound at threeconcentrations and incubated for 72 h. Probe substrates for CYP3A4 andCYP1A2 are added for 30 minutes and 1 h before the end of theincubation. At 72 h, cells and media are removed and the extent ofmetabolism of each probe substrate quantified by LC-MS/MS. Theexperiment is controlled by using inducers of the individual P450sincubated at one concentration in triplicate.

Example 89 Plasma Protein Binding

Solutions of Formula I compound (5 um, 0.5% final DMSO concentration)are prepared in buffer and 10% plasma (v/v in buffer). A 96 well HTdialysis plate is assembled so that each well is divided in two by asemi-permeable cellulose membrane. The buffer solution is added to oneside of the membrane and the plasma solution to the other side;incubations are then conducted at 37° C. over 2 h in triplicate. Thecells are subsequently emptied, and the solutions for each batch ofcompounds are combined into two groups (plasma-free andplasma-containing) then analysed by LC-MSMS using two sets ofcalibration standards for plasma-free (6 points) and plasma-containingsolutions (7 points). The fraction unbound value for the compound iscalculated.

Example 90 hERG Channel Blockage

Formula I compounds are evaluated for ability to modulate rubidiumefflux from HEK-294 cells stably expressing hERG potassium channelsusing established flux methodology. Cells are prepared in mediumcontaining RbCl, plated into 96-well plates and grown overnight to formmonolayers. The efflux experiment is initiated by aspirating the mediaand washing each well with 3×100 μL of pre-incubation buffer (containinglow [K⁺]) at room temperature. Following the final aspiration, 50 μL ofworking stock (2×) compound is added to each well and incubated at roomtemperature for 10 minutes. 50 μL of stimulation buffer (containing high[K+]) is then added to each well giving the final test compoundconcentrations. Cell plates are then incubated at room temperature for afurther 10 minutes. 80 μL of supernatant from each well is thentransferred to equivalent wells of a 96-well plate and analysed viaatomic emission spectroscopy. The compound is screened as 10 ptduplicate IC₅₀ curves, n=2, from a top concentration of 100 μM.

The foregoing description is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will be readily apparent to those skilled in the art, it is notdesired to limit the invention to the exact construction and processshown as described above. Accordingly, all suitable modifications andequivalents may be considered to fall within the scope of the inventionas defined by the claims that follow.

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, or groupsthereof.

What is claimed is:
 1. A compound selected from Formula I:

and stereoisomers, geometric isomers, tautomers, or pharmaceuticallyacceptable salts thereof, wherein: R¹ is H; R² is methyl or ethyl; R³ is1H-indazol-4-yl; R⁴ is selected from —NR¹⁰R¹³, —NR¹²C(═O)R¹⁰,—NR¹⁰C₁-C₁₂ alkyl)NR¹⁰R¹³, —NR¹⁰(C₁-C₁₂ alkyl)OR¹⁰, —NR¹⁰(C₁-C₁₂alkyl)C(═O)NR¹⁰R¹³, —NR¹⁰(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl),—NR¹⁰(C₁-C₁₂ alkylene)-(C₂-C₂₀ heterocyclyl), —NR¹⁰(C₁-C₁₂alkylene)-(C₆-C₂₀ aryl), —NR¹⁰(C₁-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl),—OR¹⁰, —O(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl), —O(C₁-C₁₂alkylene)-(C₂-C₂₀ heterocyclyl), —O(C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl),—O(C₁-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl), —(C₁-C₁₂ alkylene)NR¹⁰R¹³,—(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl), —(C₁-C₁₂ alkylene)-(C₂-C₂₀heterocyclyl), —(C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl), —(C₁-C₁₂alkylene)-(C₁-C₂₀ heteroaryl), —(C₂-C₈ alkynylene)NR¹⁰R¹³, —(C₂-C₈alkynylene)-(C₃-C₁₂ carbocyclyl), —(C₂-C₈ alkynylene)-(C₂-C₂₀heterocyclyl), —(C₂-C₈ alkynylene)-(C₆-C₂₀ aryl), —(C₂-C₈alkynylene)-(C₁-C₂₀ heteroaryl), —(C₁-C₁₂ alkylene)-(C₆-C₂₀arylene)-(C₂-C₂₀ heterocyclyl), —(C₆-C₂₀ aryl)-(C₁-C₁₂ alkylene)-(C₂-C₂₀heterocyclyl), —C(═O)NR¹⁰R¹¹, C₁-C₁₂ alkyl, C₂-C₈ alkynyl, C₃-C₁₂carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, and C₁-C₂₀ heteroaryl,where alkyl, alkylene, alkynyl, alkynylene, carbocyclyl, heterocyclyl,aryl, and heteroaryl are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂OH, —CN, —CF₃,—CO₂H, —COCH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃,—NHCOCH₃, —NHS(O)₂CH₃, —OH, —OCH₃, —S(O)₂N(CH₃)₂, —SCH₃, —CH₂OCH₃, and—S(O)₂CH₃; or wherein R⁴ is 4-(2-pyridyl)piperazin-1-yl; R¹⁰, R¹¹ andR¹² are independently selected from H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl are optionally substituted with oneor more groups independently selected from F, Cl, Br, I, —CH₂OH,—CH₂C₆H₅, —CN, —CF₃, —CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃,—NHS(O)₂CH₃, —OH, —OCH₃, —OCH₂CH₃, —S(O)₂NH₂, —SCH₃, —S(O)CH₃, —CH₂OCH₃,—CH₃, and —S(O)₂CH₃; or R¹⁰ and R¹¹ together with the nitrogen atom towhich they are attached form a C₂-C₂₀ heterocyclyl ring; and R¹³ isselected from C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, and C₁-C₂₀ heteroaryl,where alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, —SCH₃, —S(O)CH₃, —OCH₂CH₂—N(CH₃)₂, and—S(O)₂CH₃; or R¹⁰ and R¹³ together with the nitrogen atom to which theyare attached form a C₂-C₂₀ heterocyclyl ring.
 2. The compound of claim 1wherein R² is CH₃.
 3. The compound of claim 1 wherein R⁴ is optionallysubstituted pyrimidin-5-yl.
 4. The compound of claim 1 wherein R⁴ isoptionally substituted C₆-C₂₀ aryl.
 5. The compound of claim 4 whereinR⁴ is optionally substituted phenyl.
 6. The compound of claim 1 whereinR⁴ is —OR¹⁰ where R¹⁰ is optionally substituted phenyl.
 7. The compoundof claim 6 wherein phenyl is substituted with one or more groupsindependently selected from —OCH₃, —SO₂CH₃, —SO₂NH₂, —NHSO₂CH₃,—CH₂OCH₃, —CN, —C(═O)NH₂, —C(═O)NHCH₃, —NHC(═O)CH₃, —CF₃, —OH, —CH₃, and—Cl.
 8. The compound of claim 1 wherein R⁴ is —OR¹⁰ where R¹⁰ isoptionally substituted pyridyl.
 9. The compound of claim 1 wherein R⁴ is—OR¹⁰ where R¹⁰ is optionally substituted C₁-C₁₂ alkyl.
 10. The compoundof claim 1 wherein R⁴ is —O(C₁-C₁₂ alkylene)-(C₂-C₂₀ heterocyclyl),—O(C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl), or —O(C₁-C₁₂ alkylene)-(C₁-C₂₀heteroaryl).
 11. The compound of claim 1 wherein R⁴ is —NR¹⁰R¹³ whereR¹⁰ is H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, or C₂-C₈ alkynyl; and R¹³ isoptionally substituted phenyl.
 12. The compound of claim 1 wherein R⁴ is—NR¹⁰R¹³ where R¹⁰ is H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, or C₂-C₈ alkynyl;and R¹³ is optionally substituted indazol-6-yl or indazol-4-yl.
 13. Thecompound of claim 1 wherein R⁴ is —NR¹⁰(C₁-C₁₂ alkyl)NR¹⁰R¹³,—NR¹⁰(C₁-C₁₂ alkyl)OR¹⁰, or —NR¹⁰(C₁-C₁₂ alkyl)C(═O)NR¹⁰R¹³.
 14. Thecompound of claim 1 wherein R⁴ is morpholinyl, 4-methylpiperazin-1-yl,4-methylsulfonylpiperazin-1-yl, or 4-(2-pyridyl)piperazin-1-yl.
 15. Acompound selected from:4-(3,4-dimethoxyphenoxy)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)phenoxy)-1H-pyrazolo[3,4-d]pyrimidine;N-(3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)methanesulfonamide;6-(1H-indazol-4-yl)-4-(3-(methoxymethyl)phenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)benzonitrile;3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N-methylbenzamide;6-(1H-indazol-4-yl)-4-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;N-(3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)phenyl)acetamide;6-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-4-(4-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;4-(3,4-dimethoxyphenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(pyridin-3-yloxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-4-(3-methoxyphenoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-N-(3-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(3-(methylsulfonyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(pyridin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-N-(2-methoxyethyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;N1-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N1,N2,N2-trimethylethane-1,2-diamine;N-(3,4-dimethoxyphenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-3-yl)ethoxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)benzyloxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(3-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine;4-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzamide;4-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzenesulfonamide;4-(benzo[d][1,3]dioxol-5-yl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;4-(3,4-dichlorophenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-N-(4-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(2-phenoxyethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(2-(pyridin-3-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-4-(3-(trifluoromethyl)phenethoxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-phenethoxy-1H-pyrazolo[3,4-d]pyrimidine;1-ethyl-6-(1H-indazol-4-yl)-N-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-4-(4-methylpiperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(4-(methylsulfonyl)piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine;4-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-S-dioxothiomorpholine;6-(1H-indazol-4-yl)-4-(2-methoxyethoxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;4-(benzyloxy)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-4-(3-methoxybenzyloxy)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(2-morpholinoethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;N-(benzo[d][1,3]dioxol-5-yl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;N-(4-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)acetamide;6-(1H-indazol-4-yl)-1-methyl-4-(3-(pyridin-3-yl)propoxy)-1H-pyrazolo[3,4-d]pyrimidine;3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N,N-dimethylbenzamide;6-(1H-indazol-4-yl)-1-methyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;N-(4-(2-(dimethylamino)ethoxy)phenyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-(3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)propyl)pyrrolidin-2-one;4-(2-(1H-imidazol-1-yl)ethoxy)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(pyridin-4-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(3-(pyridin-4-yl)propoxy)-1H-pyrazolo[3,4-d]pyrimidine;1-(2-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)ethyl)pyrrolidin-2-one;6-(1H-indazol-4-yl)-1-methyl-N-(3-(pyrrolidin-1-yl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;N1-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N3,N3-dimethylpropane-1,3-diamine;6-(1H-indazol-4-yl)-1-methyl-N-(3-morpholinopropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-2-yl)ethoxy)-1H-pyrazolo[3,4-d]pyrimidine;N-(3-(1H-imidazol-1-yl)propyl)-6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-4-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(2-(pyridin-4-yl)ethoxy)-1H-pyrazolo[3,4-d]pyrimidine;1-(4-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)piperidin-1-yl)ethanone;6-(1H-indazol-4-yl)-1-methyl-4-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine;3-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)-1-(piperazin-1-yl)propan-1-one;6-(1H-indazol-4-yl)-1-methyl-4-(2-(4-(methylsulfonyl)piperazin-1-yl)ethoxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(3-((4-(methylsulfonyl)piperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(4-((4-(methylsulfonyl)piperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-N-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(3-(methylsulfonyl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-4-(2-(piperazin-1-yl)ethoxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-4-(piperidin-4-yloxy)-1H-pyrazolo[3,4-d]pyrimidine;6-(1H-indazol-4-yl)-1-methyl-N-(3-(methylsulfinyl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(1H-indazol-4-yl)-1-methyl-N-(3-(methylthio)propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;and4-(2-(6-(1H-indazol-4-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)ethyl)morpholine.16. A pharmaceutical composition comprised of a compound of claim 1 anda pharmaceutically acceptable carrier, glidant, diluent, or excipient.17. The pharmaceutical composition according to claim 16, furthercomprising an additional therapeutic agent selected from achemotherapeutic agent, an anti-inflammatory agent, an immunomodulatoryagent, a neurotropic factor, an agent for treating cardiovasculardisease, an agent for treating liver disease, an anti-viral agent, anagent for treating blood disorders, an agent for treating diabetes, andan agent for treating immunodeficiency disorders.
 18. A process formaking a pharmaceutical composition which comprises combining a compoundof claim 1 with a pharmaceutically acceptable carrier.
 19. A method ofmaking a Formula I compound as described in claim 1 comprising reactinga Formula II compound:

with a boronate compound whereby the Formula I compound is formed.
 20. Apharmaceutical composition comprised of a compound of claim 15 and apharmaceutically acceptable carrier, glidant, diluent, or excipient. 21.The pharmaceutical composition according to claim 20, further comprisingan additional therapeutic agent selected from a chemotherapeutic agent,an anti-inflammatory agent, an immunomodulatory agent, a neurotropicfactor, an agent for treating cardiovascular disease, an agent fortreating liver disease, an anti-viral agent, an agent for treating blooddisorders, an agent for treating diabetes, and an agent for treatingimmunodeficiency disorders.
 22. A kit for treating a PI3K-mediatedcondition, comprising: a) a first pharmaceutical composition comprisinga compound of claim 15; and b) instructions for use.
 23. The compound ofclaim 1 wherein R⁴ is selected from —NR¹⁰R¹³, —NR¹²C(═O)R¹⁰,—NR¹⁰(C₁-C₁₂ alkyl)NR¹⁰R¹³, —NR¹⁰(C₁-C₁₂ alkyl)OR¹⁰, —NR¹⁰(C₁-C₁₂alkyl)C(═O)NR¹⁰R¹³, —NR¹⁰(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl),—NR¹⁰(C₁-C₁₂ alkylene)-(C₂-C₂₀ heterocyclyl), —NR¹⁰(C₁-C₁₂alkylene)-(C₆-C₂₀ aryl), —NR¹⁰(C₁-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl),—OR¹⁰, —O(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl), —O(C₁-C₁₂alkylene)-(C₂-C₂₀ heterocyclyl), —O(C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl),—O(C₁-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl), —(C₁-C₁₂ alkylene)NR¹⁰R¹³,—(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl), —(C₁-C₁₂ alkylene)-(C₂-C₂₀heterocyclyl), —(C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl), —(C₁-C₁₂alkylene)-(C₁-C₂₀ heteroaryl), —(C₂-C₈ alkynylene)NR¹⁰R¹³, —(C₂-C₈alkynylene)-(C₃-C₁₂ carbocyclyl), —(C₂-C₈ alkynylene)-(C₂-C₂₀heterocyclyl), —(C₂-C₈ alkynylene)-(C₆-C₂₀ aryl), —(C₂-C₈alkynylene)-(C₁-C₂₀ heteroaryl), —(C₁-C₁₂ alkylene)-(C₆-C₂₀arylene)-(C₂-C₂₀ heterocyclyl), —(C₆-C₂₀ aryl)-(C₁-C₁₂ alkylene)-(C₂-C₂₀heterocyclyl), —C(═O)NR¹⁰R¹¹, C₁-C₁₂ alkyl, C₂-C₈ alkynyl, C₃-C₁₂carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, and C₁-C₂₀ heteroaryl,where alkyl, alkylene, alkynyl, alkynylene, carbocyclyl, heterocyclyl,aryl, and heteroaryl are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂OH, —CN, —CF₃,—CO₂H, —COCH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃,—NHCOCH₃, —NHS(O)₂CH₃, —OH, —OCH₃, —S(O)₂N(CH₃)₂, —SCH₃, —CH₂OCH₃, and—S(O)₂CH₃.